Prodigiosin Analogs And Methods Of Use

ABSTRACT

Prodigiosin analogs which reactivate the p53 pathway are provided, as well as compositions of these compounds, and methods for reactivation of the p53 pathway using these compounds are provided. The prodigiosin analogs may be used to treat cancer in which p53 mutation plays a role, including prostate cancer, breast cancer, kidney cancer, ovarian cancer, lymphoma, leukemia, and glioblastoma, among others.

REFERENCE TO GOVERNMENT GRANTS

This invention was made with government support under Grant No. CA176289awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD

The present disclosure relates generally to the field of formulationchemistry. More particularly, the present disclosure relates tocompounds, compositions, and methods for treating cancer, specificallyby restoring the p53 pathway and inducing the expression of the p73protein.

BACKGROUND

Prodigiosin (represented by tautomeric Formulas (A1) and (A2)) is theparent member of the tripyrrole alkaloid family of natural products thatshows potent anti-cancer activity against tumors with mutated p53proteins.

Activation of p53 can induce cell-cycle arrest and apoptosis throughtranscriptional regulation of specific target genes. However, p53 ismutated in more than 50% of tumors, making functional reactivation ofthe p53 pathway an attractive strategy for cancer therapy development.Prodigiosin is further able to induce the expression of the p73 proteinand disrupt its interaction with mutant p53, thereby rescuing p53pathway deficiency and promoting anti-tumor effects. Accordingly, it isdesirable to identify and synthesize prodigiosin analogs that aresuitable as cancer treatments through restoration of the p53 pathway andinducing the expression of the p73 protein.

SUMMARY

The present disclosure provides compounds of Formula XIV:

wherein: R¹ and R² are, independently, selected from the groupconsisting of H, OH, halogen, —C₁₋₆alkyl, —C₁₋₆fluoroalkyl, —CN, —NO₂,—OR⁷, —SR⁷, —S(═O)R⁷, —S(═O)₂R⁷, —NHS(═O)₂R⁷, —C(═O)R⁷, —OC(═O)R⁷,—CO₂R⁷, —OCO₂R⁷, —CH(R⁷)₂, —N(R⁷)₂, —C(═O)N(R⁷)₂, —NHC(═O)NHR⁷,—NHC(═O)R⁷, —NHC(═O)OR⁷, —C(OH)(R⁷)₂, and —C(NH₂)(R⁷)₂; each R⁷ is,independently, H, halogen, or C₁-C₆alkyl, wherein the alkyl group isoptionally substituted by 1, 2, 3, 4, or 5 substituents independentlyselected from halogen, OH, CN, and NO₂; R³ is an optionally substitutedaryl or an optionally substituted heteroaryl; R⁴, R⁵, and R⁶ are,independently, OH, —C₁₋₁₀alkyl, —OC₁₋₁₀alkyl, or —SC₁₋₁₀alkyl, whereineach alkyl group is, independently, optionally substituted by 1, 2, 3,4, or 5 substituents independently selected from halogen, OH, CN, andNO₂; and n is an integer from 0 to 5; or an isomer, tautomer, or solvatethereof, or a pharmaceutically acceptable salt thereof; provided that:if n is 0, then R³ is not an optionally substituted pyrrolyl; and if nis 2, then the compound is not

The present disclosure also provides pharmaceutical compositionscomprising the compounds, or an isomer, tautomer, or solvate thereof, ora pharmaceutically acceptable salt thereof, of Formula XIV and apharmaceutically acceptable carrier.

The present disclosure also provides methods of treating cancer in asubject comprising administering to the subject in need thereof thecompound, or isomer, tautomer, or solvate thereof, or pharmaceuticallyacceptable salt thereof, of Formula XIV.

The present disclosure also provides methods of preparing a compound ofFormula XIV:

wherein: R¹ and R² are, independently, selected from the groupconsisting of H, OH, halogen, —C₁₋₆alkyl, —C₁₋₆fluoroalkyl, —CN, —NO₂,—SR⁷, —S(═O)R⁷, —S(═O)₂R⁷, —NHS(═O)₂R⁷, —C(═O)R⁷, —OC(═O)R⁷, —CO₂R⁷,—OCO₂R⁷, —CH(R⁷)₂, —N(R⁷)₂, —C(═O)N(R⁷)₂, —NHC(═O)NHR⁷, —NHC(═O)R⁷,—NHC(═O)OR⁷, —C(OH)(R⁷)₂, and —C(NH₂)(R⁷)₂; each R⁷ is, independently,H, halogen, or C₁-C₆alkyl, wherein the alkyl group is optionallysubstituted by 1, 2, 3, 4, or 5 substituents independently selected fromhalogen, OH, CN, and NO₂; R³ is an optionally substituted aryl or anoptionally substituted heteroaryl; R⁴, R⁵, and R⁶ are, independently,—OH, —OC₁₋₁₀alkyl, or —SC₁₋₁₀alkyl, wherein each alkyl group is,independently, optionally substituted by 1, 2, 3, 4, or 5 substituentsindependently selected from halogen, OH, CN, and NO₂; and n is aninteger from 0 to 5; wherein the method comprises: admixing a solvent,an acid, a compound of Formula XII

and a compound of Formula XIII

at a temperature sufficient to result in the formation of the compoundof Formula XIV; the acid is selected from the group consisting ofhydrochloric acid, hydrobromic acid, hydrofluoric acid, acetic acid,phosphoric acid, toluenesulfonic acid, sulfuric acid, and nitric acid;and the temperature is about from about 0° C. to about 100° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts various aspects of a p53-responsive Luciferase ReporterAssay experiment conducted using prodigiosin analogs.

FIGS. 2A and 2B depict various aspects of a p53-responsive LuciferaseReporter Assay experiment conducted using prodigiosin analogs.

FIG. 3 depicts various aspects of a western blotting experimentconducted using prodigiosin analogs.

FIG. 4 depicts various aspects of a western blotting experimentconducted using prodigiosin analogs.

FIG. 5 depicts various aspects of a western blotting experimentconducted using prodigiosin analogs.

FIG. 6 depicts various aspects of a western blotting experimentconducted using prodigiosin analogs.

FIG. 7 depicts various aspects of a western blotting experimentconducted using prodigiosin analogs.

FIG. 8 depicts various aspects of a western blotting experimentconducted using prodigiosin analogs.

FIG. 9 depicts various aspects of a western blotting experimentconducted using prodigiosin analogs.

FIG. 10 depicts various aspects of a western blotting experimentconducted using prodigiosin analogs.

FIG. 11 depicts various aspects of a CellTiter-Glo luminescent cellviability assay experiment conducted using prodigiosin analogs.

FIG. 12 depicts various aspects of a CellTiter-Glo luminescent cellviability assay experiment conducted using prodigiosin analogs.

FIG. 13 depicts various aspects of a CellTiter-Glo luminescent cellviability assay experiment conducted using prodigiosin analogs.

FIG. 14 depicts various aspects of a CellTiter-Glo luminescent cellviability assay experiment conducted using prodigiosin analogs.

FIG. 15 depicts various aspects of a CellTiter-Glo luminescent cellviability assay experiment conducted using prodigiosin analogs.

FIG. 16 depicts various aspects of a CellTiter-Glo luminescent cellviability assay experiment conducted using prodigiosin analogs.

FIG. 17 depicts various aspects of a flow cytometry assay experimentconducted using prodigiosin analogs.

FIG. 18 depicts various aspects of a flow cytometry assay experimentconducted using prodigiosin analogs.

FIG. 19 depicts various aspects of a flow cytometry assay experimentconducted using prodigiosin analogs.

FIG. 20 depicts various aspects of a colony formation assay experimentconducted using prodigiosin analogs.

FIG. 21 depicts various aspects of a colony formation assay experimentconducted using prodigiosin analogs.

FIG. 22 depicts various aspects of an immunofluorescence experimentconducted using prodigiosin analogs.

FIG. 23 depicts various aspects of an immunofluorescence experimentconducted using prodigiosin analogs.

FIG. 24 depicts various aspects of an immunofluorescence experimentconducted using prodigiosin analogs.

FIG. 25 depicts various aspects of an immunofluorescence experimentconducted using prodigiosin analogs.

FIGS. 26A, 26B, 26C, 26D, and 26E depict PG3-Oc inhibition of the growthof p53-mutant cancer cell lines.

FIGS. 27A, 27B, 27C, 27D, and 27E depict PG3-Oc induction of apoptosisin p53 mutant cancer cell lines, Caspase 3/7 activity assay, HT29 cellsco-treated with 1 μM PG3-Oc and pan-caspase inhibitor Z-VAD-fmk, andWestern blotting analysis of active caspase-8, active caspase-3 andcleaved PARP in HT29 cells and SW480 cells.

FIGS. 28A, 28B, 28C, and 28D depict PG3-Oc restoration of p53 pathway inp53 mutant cancer cell lines.

FIGS. 29A, 29B, 29C, 29D, and 29E depict the induction of PUMA iscorrelated with cell death.

FIGS. 30A, 30B, 30C, 30D, 30E, 30F, 30G, and 30H depict that PUMA is akey effector of PG3-Oc-mediated apoptosis in mutant p53 cell lines.

FIGS. 31A, 31B, 31C, 31D, and 31E depict the exploration of themolecular mechanism of PG3-Oc-induced up-regulation of PUMA.

FIGS. 32A and 32B depict time-course analysis of caspase activation.

FIGS. 33A, 33B, 33C, and 33D depict the exploration of the molecularmechanism of PG3-Oc-induced up-regulation of PUMA.

FIGS. 34A, 34B, 34C, 34D, 34E, 34F, 34G, 34H, and 34I depict knock-outof PUMA by CRISPR/Cas9 gene editing.

FIG. 35 depicts a representative synthetic scheme for PG3-Oc.

FIG. 36 depicts Mass spectrum analysis of Compound 3 in FIG. 35.

FIG. 37 depicts Mass spectrum analysis of PG3-Oc.

FIG. 38 depicts ¹H NMR analysis of PG3-Oc.

FIG. 39 shows progigiosin and PG3-Oc structures and a representativesynthetic preparation of PG3-Oc.

FIGS. 40 and 41 show cell viability assay, dose response curves, andIC50 value measurement of PG3-Oc in a panel of cancer cell lines.

FIG. 42 shows HT29 cells treated with PG3-Oc.

FIG. 43 shows that PG3-Oc restores p53 target gene expression.

FIG. 44 shows the GSEA plot: Representative gene set from 1867differential expression genes showing specific responses to the p53pathway.

FIG. 45 shows cells treated with 1 μM PG3-Oc for 8 and 18 h.

FIG. 46 shows fluorescence microscopy localization of PG3-Oc andprodigiosin in HT29 and SW480 cells.

FIG. 47 shows PG3-Oc did not induce DNA damage.

FIG. 48 shows immunofluorescence staining for γ-H2AX foci.

FIG. 49 shows HT29 cells were transfected with Control, PUMA, DR5 andPUMA/DR5 siRNAs.

FIG. 50 shows cell death analyzed by nuclear PI-staining using flowcytometry.

FIG. 51 shows cleavage of caspases and PARP were detected by westernblotting using the indicated antibodies.

FIG. 52 shows sub G1 populations analyzed by flow cytometry.

FIG. 53 shows HCT116 p53−/− cells transfected with the indicated siRNAs.

FIG. 54 shows HT29 cell western blots performed using the indicatedantibodies.

FIG. 55 shows HT29 cells transfected with the indicated siRNAs.

FIG. 56 shows HT29 cells transfected with the indicated siRNAs.

FIG. 57 shows HT29 and HT29-PUMA-KO cell western blots using theindicated antibodies.

FIG. 58 shows HT29 and HT29-PUMA-KO cells transfected with indicatedsiRNAs.

FIG. 59 shows suggested model of PG3-Oc induced upregulation of PUMA andDR5.

FIG. 60 shows various mutant p53-expressing cancer cell lines treatedwith PG3-Oc for 24 hours.

FIG. 61 shows HT29 cells treated with 1 μM PG3-Oc for 24 h.

FIG. 62 shows Jurkat cells treated with PG3-Oc at the indicated dosesand time points.

FIG. 63 shows HCT116 and HCT116 p53−/− cells treated with PG3-Oc at theindicated doses for 24 h.

FIG. 64 shows cells transfected with c-Myc siRNAs and control siRNAs.

FIG. 65 shows mRNAs extracted for qRT-PCR analysis.

FIG. 66 shows HT29 cells transfected with the vector pcDNA3 andpcDNA3-cMyc.

FIG. 67 shows mRNA extracted for qRT-PCR analysis.

FIG. 68 shows HT29 and HCT115 p53−/− cells treated with PG3-Oc for 18hours.

FIG. 69 shows cells co-treated with the proteasome inhibitor MG132.

FIG. 70 shows analysis of c-Myc target genes from RNA-Seq analysis withdifferential gene expression induced by 1 μM PG3-Oc in HT29 cells.

FIG. 71 shows a GSEA plot.

FIG. 72 shows HT29 cells treated with 1 μM PG3-Oc for the indicated timepoints.

FIG. 73 shows HT29 cells treated with PG3-Oc, SCH772984 or U0126respectively, or co-treatment with PG3-Oc/SCH772984 or PG3-Oc/U0126.

FIG. 74 shows HT29 cells pre-treated with PG3-Oc, SCH772984 orgefitinib.

FIG. 75 shows HT29 cells transfected with control siRNA.

FIG. 76 shows HT29 cells treated with SCH772984.

FIG. 77 shows HT29 and HCT116 p53−/− cells treated with 4 μM PG3-Oc andregorafenib.

FIG. 78 shows the relative tumor growth is normalized tumor size to thetumor size of day 1 before the treatment (*, p<0.05 by an unpaired ttest).

FIG. 79 shows the mean tumor volume before and after treatment (*,p<0.05 by an unpaired t test).

FIG. 80 shows images of 5 representative tumors from vehicle control andtreated groups.

FIG. 81 shows body weight changes of nude mice during treatment period(*, p<0.05 by an unpaired t test).

FIG. 82 shows Ki-67 and PUMA antibody staining.

FIG. 83 shows the proposed mechanism of PG3-Oc-induced upregulation ofPUMA through ERK1/2/c-Myc/PUMA pathway, and upregulation of DR5 throughATF4/CHOP axis.

FIGS. 84 and 85 show colony formation assays of p53-mutant and p53-nullhuman cancer cells.

FIG. 86 shows cell-cycle profiles after PG3-Oc treatment.

FIG. 87 shows cell-cycle profiles after PG3-Oc treatment and apoptosisanalyzed by nuclear PI-staining using flow cytometry.

FIG. 88 shows a heat-map depicting differential gene expression of genesin the p53 pathway in PG3-Oc treated cells as identified by IPAanalysis.

FIG. 89 shows a subset of p53 negatively regulated target genes, besidesSCD, was significantly downregulated by PG3-Oc.

FIG. 90 shows a subset of p53 positively regulated target genes, besidesHSPA4L, DDB2 and PIDD1, was significantly upregulated by PG3-Oc.

FIG. 91 shows HT29 and HT29-PUMA-KO cells treated with PG3-Oc orco-treated with caspase 8 inhibitor (cas8 inh) and pan-caspase(Z-VAD-FMK) inhibitor for 48 hours.

FIG. 92 shows HEK293 cells transfected to the control dish.

FIG. 93 shows HT29 cells transfected with p73 and p63 siRNAs.

FIG. 94 shows HT29 cells treated with 1 μM PG3-Oc or co-treated withSB216763.

FIG. 95 shows the Western blot analysis of the expression of PUMAprotein from single cell colonies isolated from a pool of HT29-PUMA-KOcells.

FIG. 96 shows the Western blot analysis of the expression of PUMAprotein from single cell colonies isolated from a pool of HT29-PUMA-KOcells.

FIG. 97 shows PG3-Oc co-treatment with SB216763.

FIG. 98 shows cells treated with PG3-Oc for 24 hours.

FIG. 99 shows cells transfected with control siRNA or GSK3β siRNA.

FIG. 100 shows cells transfected with control siRNA or GSK3α/β siRNA.

DESCRIPTION OF EMBODIMENTS

Various terms relating to aspects of the present disclosure are usedthroughout the specification and claims. Such terms are to be giventheir ordinary meaning in the art, unless otherwise indicated. Otherspecifically defined terms are to be construed in a manner consistentwith the definition provided herein. Unless defined otherwise, alltechnical and scientific terms have the same meaning as is commonlyunderstood by one of ordinary skill in the art to which the disclosedembodiments belong.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless expressly stated otherwise.

As used herein, the term “about” means that the recited numerical valueis approximate and small variations would not significantly affect thepractice of the disclosed embodiments. Where a numerical value is used,unless indicated otherwise by the context, “about” means the numericalvalue can vary by ±10% and remain within the scope of the disclosedembodiments.

As used herein, the term “alkoxy” means a straight or branched —O-alkylgroup having 1 to 20 carbon atoms. In some embodiments, the alkoxy grouphas from 1 to 10 carbon atoms, from 1 to 8 carbon atoms, from 1 to 6carbon atoms, from 1 to 4 carbon atoms, from 2 to 10 carbon atoms, from2 to 8 carbon atoms, from 2 to 6 carbon atoms, or from 2 to 4 carbonatoms. Examples of alkoxy groups include, but are not limited to,methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, and the like.

As used herein, “alkyl” refers to a saturated straight or branchedhydrocarbon, such as a straight or branched group of 1-12, 1-10, or 1-6carbon atoms, referred to herein as C₁-C₁₂alkyl, C₁-C₁₀alkyl, andC₁-C₆alkyl, respectively. Exemplary alkyl groups include, but are notlimited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl,2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl,2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl,4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl,2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,hexyl, heptyl, octyl, etc. An alkyl group can be substituted (i.e.,optionally substituted) with one or more substituents or can bemulticyclic as set forth below.

As used herein, “ether” and “ether group” refer to a functional groupcomprising two hydrocarbon groups covalently linked by an oxygen.

As used herein, the term “amino” means —NH2.

As used herein, “ring structure” includes aryl, cycloalkyl, heteroaryl,and heterocyclyl.

As used herein, “aryl” is art-recognized and refers to a carbocyclicaromatic group. Representative aryl groups include, but are not limitedto, phenyl, naphthyl, anthracenyl, and the like. Unless specifiedotherwise, the aromatic ring may be substituted at one or more ringpositions with, for example, halogen, azide, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino,amido, carboxylic acid, —C(O)alkyl, —CO₂alkyl, carbonyl, carboxyl,alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester,heterocyclyl, heteroaryl, —CF₃, —CN, or the like. “Aryl” also includespolycyclic ring systems having two or more carbocyclic rings in whichtwo or more carbons are common to two adjoining rings (the rings are“fused rings”) wherein at least one of the rings is aromatic, and theother ring(s) may be, for example, cycloalkyl, cycloalkenyl,cycloalkynyl, and/or aryl. “Haloaryl” refers to an aryl group that issubstituted with at least one halogen. In some embodiments, the aromaticgroup is not substituted (i.e., it is unsubstituted).

As used herein, “cycloalkyl” means a non-aromatic mono- or multi-cyclicring system of about 3 to about 10 carbon atoms, or about 5 to about 10carbon atoms. Suitable cycloalkyl rings contain about 5 to about 6 ringatoms. The cycloalkyl can be optionally substituted with one or more“ring system substituents” which may be the same or different, and areas defined herein. Representative monocyclic cycloalkyls include, butare not limited to, cyclopentyl, cyclohexyl, cycloheptyl, and the like.Representative multicyclic cycloalkyl include, but are not limited to,1-decalin, norbornyl, adamantyl, and the like. In such cycloalkyl groupsand, including the C₅-C₇ cycloalkyl groups, one or two of the carbonatoms forming the ring can optionally be replaced with a hetero atom,such as sulfur, oxygen or nitrogen. Examples of such groups include, butare not limited to, piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl,imidazolidinyl, oxazolidinyl, perhydroazepinyl, perhydrooxazapinyl,oxepanyl, perhydrooxepanyl, tetrahydrofuranyl, and tetrahydropyranyl. C₃and C₄ cycloalkyl groups having a member replaced by nitrogen or oxygeninclude, but are not limited to, aziridinyl, azetidinyl, oxetanyl, andoxiranyl.

As used herein, “heteroaryl” is art-recognized and refers to aromaticgroups that include at least one ring heteroatom. In some embodiments, aheteroaryl group contains 1, 2, 3, or 4 ring heteroatoms. Representativeexamples of heteroaryl groups includes, but are not limited to, pyridyl,pyrimidinyl, pyrazinyl, pyridazinyl, pyridinyl (including2-aminopyridine), triazinyl, furyl, quinolyl, isoquinolyl, thienyl,imidazolyl, thiazolyl, indolyl (such as indol-3-yl), pyrryl, oxazolyl,benzofuryl, benzothienyl, pyrazolyl, benzthiazolyl, isoxazolyl,triazolyl (including 1,2,4-triazole, 1,2,3-triazole, and5-amino-1,2,4-triazole), tetrazolyl, indazolyl, isothiazolyl,1,2,4-thiadiazolyl, benzothienyl, purinyl, carbazolyl, isoxazolyl,benzimidazolyl, indolinyl, pyranyl, pyrazolyl, triazolyl, oxadiazolyl(including 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole,3-amino-1,2,4-oxadiazole, 1,3,4-oxadiazole), thianthrenyl, indolizinyl,isoindolyl, isobenzofuranyl, pyrrolyl, benzoxazolyl, xanthenyl,2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, phthalazinyl, acridinyl,naphthyridinyl, quinazolinyl, phenanthridinyl, perimidinyl,phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl,furazanyl, phenoxazinyl groups, and the like. Unless specifiedotherwise, the heteroaryl ring may be substituted at one or more ringpositions with, for example, halogen, azide, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino,amido, carboxylic acid, —C(O)alkyl, —CO₂alkyl, carbonyl, carboxyl,alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester,heterocyclyl, aryl, —CF₃, —CN, or the like. “Heteroaryl” also includespolycyclic ring systems having two or more rings in which two or morecarbons are common to two adjoining rings (the rings are “fused rings”)wherein at least one of the rings is heteroaromatic, and the otherring(s) may be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl,and/or aryl.

As used herein, “heterocyclyl” and “heterocyclic group” areart-recognized and refer to saturated, partially unsaturated, oraromatic 3- to 10-membered ring structures, alternatively 3- to7-membered rings, whose ring structures include one to four heteroatoms,such as nitrogen, oxygen, and sulfur. Heterocycles may also be mono-,bi-, or other multi-cyclic ring systems. A heterocycle may be fused toone or more aryl, partially unsaturated, or saturated rings.Heterocyclyl groups include, but are not limited to, biotinyl,chromenyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dihydrothienyl,dithiazolyl, homopiperidinyl, imidazolidinyl, isoquinolyl,isothiazolidinyl, isoxazolidinyl, morpholinyl, oxolanyl, oxazolidinyl,phenoxanthenyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl,pyrazolinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolidin-2-onyl,pyrrolinyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydropyranyl,tetrahydroquinolyl, thiazolidinyl, thiolanyl, thiomorpholinyl,thiopyranyl, xanthenyl, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. Unless specifiedotherwise, the heterocyclic ring is optionally substituted at one ormore positions with substituents such as alkanoyl, alkoxy, alkyl,alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido,carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl,halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone,nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide,sulfonamido, sulfonyl and thiocarbonyl. In some embodiments, theheterocyclyl group is not substituted (i.e., it is unsubstituted).

As used herein, “in need thereof” means that the “individual,”“subject,” or “patient” has been identified as having a need for theparticular method, prevention, or treatment. In some embodiments, theidentification can be by any means of diagnosis. In any of the methods,preventions, and treatments described herein, the “individual,”“subject,” or “patient” can be in need thereof.

As used herein, “subject” and “patient” are used interchangeably. Asubject may be any animal, including mammals such as companion animals,laboratory animals, and non-human primates. In some embodiments, thesubject is a human.

As used herein, the terms “treat,” “treated,” or “treating” mean boththerapeutic treatment and prophylactic or preventative measures whereinthe object is to prevent or slow down (lessen) an undesiredphysiological condition, disorder or disease, or obtain beneficial ordesired clinical results. For purposes herein, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms; diminishment of extent of condition, disorder or disease;stabilized (i.e., not worsening) state of condition, disorder ordisease; delay in onset or slowing of condition, disorder or diseaseprogression; amelioration of the condition, disorder or disease state orremission (whether partial or total), whether detectable orundetectable; an amelioration of at least one measurable physicalparameter, not necessarily discernible by the patient; or enhancement orimprovement of condition, disorder or disease. Treatment includeseliciting a clinically significant response, optionally withoutexcessive levels of side effects. Treatment also includes prolongingsurvival as compared to expected survival if not receiving treatment.

As used herein, the term, “compound” means all stereoisomers, tautomers,isotopes, and polymorphs of the compounds described herein.

As used herein, the terms “comprising” (and any form of comprising, suchas “comprise”, “comprises”, and “comprised”), “having” (and any form ofhaving, such as “have” and “has”), “including” (and any form ofincluding, such as “includes” and “include”), or “containing” (and anyform of containing, such as “contains” and “contain”), are inclusive andopen-ended and include the options following the terms, and do notexclude additional, unrecited elements or method steps.

As used herein, the term “halo” means halogen groups and includes, butis not limited to, fluoro, chloro, bromo, and iodo.

As used herein, the term “haloalkyl” means a C₁₋₆alkyl group having oneor more halogen substituents. Examples of haloalkyl groups include, butare not limited to, —CF₃, —C₂F₅, —CHF₂, —CCl₃, —CHCl₂, —C₂Cl₅, —CH₂CF₃,and the like.

As used herein, the term “integer” means a numerical value that is awhole number. For example, an “integer from 1 to 5” means 1, 2, 3, 4, or5.

As used used herein, the phrase “optionally substituted” means that asubstitution is optional and, therefore, includes both unsubstituted andsubstituted atoms and moieties. A “substituted” atom or moiety indicatesthat any hydrogen atom on the designated compound or moiety can bereplaced with a selection from the indicated substituent groups,provided that the normal valency of the designated compound or moiety isnot exceeded, and that the substitution results in a stable compound.For example, if a methyl group is optionally substituted, then 1, 2, or3 hydrogen atoms on the carbon atom within the methyl group can bereplaced with 1, 2, or 3 of the recited substituent groups.

The compounds described herein also include hydrates and solvates, aswell as anhydrous and non-solvated forms.

Embodiments of the present disclosure include prodigiosin analogs whichhave anti-cancer activity against tumors with mutated p53 proteins.Without intending to be bound to any particular theory or mechanism ofaction, it is believed that the prodigiosin analogs result in functionalreactivation of the p53 pathway in cells with mutated p53 proteins aswell as induced expression of the p73 protein and disruption of theinteraction between p73 and mutant p53.

Prodigiosin analogs have been developed including various side groups onthe three rings of the Prodigiosin core molecule: the A-ring, theB-ring, and the C-ring, as depicted in Formula (B).

Prodigiosin analogs may include side groups attached to the coremolecule at positions A¹, A², or A³ of the A-Ring, B¹ or B² of theB-Ring, and C¹, C², or C³ of the C-Ring. Embodiments of the presentdisclosure include prodigiosin analogs with side groups on at least theC-ring of the prodigiosin core molecule, particularly at position C². Insome embodiments, the side group includes a carbonyl group. In someembodiments, the carbonyl side group is an ethyl ester (CH₂CH₂COR) or anethyl secondary amide (CH₂CH₂CONHR).

In some embodiments, the prodigiosin analog has the structure of Formula(I) or Formula (II)

Formula (I) and Formula (II) are prodigiosin analogs according toFormula (B) wherein C² is COR¹ and CH₂R², respectively. Formula (I) andFormula (II) may also be represented by tautomeric Formulas (Ia) and(IIa), respectively.

Further prodigiosin analogs according to embodiments of the disclosureare described with respect to Formula (I) and Formula (II). However, oneof ordinary skill in the art will understand that each analog could alsobe expressed as a form of Formula (Ia) or Formula (IIa), the respectivetautomers of Formula (I) and Formula (II).

A¹, A², and A³ in Formulas (I) and (II) are, independently, hydrogen,phenyl, C₁-C₂₀ alkyl or C₂-C₂₀ alkenyl, wherein the alkyl and alkenylgroups are unsubstituted or substituted by 1 to 3 substituents chosen,independently, from halogen, C₁-C₆ alkoxy, hydroxy, aryl, and aryloxy.In some embodiments, A¹, A², and A³ are hydrogen. In some embodiments,B¹ is hydrogen, C₁-C₆ alkyl, cyano, carboxy or (C₁-C₆ alkoxy) carbonyl.In some embodiments, B² is halogen, hydroxy or C₁-C₁₁ alkoxyunsubstituted or substituted by phenyl. In some embodiments, B¹ ishydrogen and B² is methoxy. In some embodiments, C¹ and C³ are,independently, hydrogen, phenyl, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀alkynyl, or C₁-C₂₀ alkoxy. In some embodiments, C¹ and C³ are methyl.

In some embodiments, the prodigiosin analog has the structure of Formula(III) or Formula (IV)

Formula (III) and Formula (IV) are Formula (I) and Formula (II),respectively, where A¹, A², A³, and B¹ are hydrogen, B² is methoxy, andC¹ and C³ are methyl.

In some embodiments, the prodigiosin analog has the structure of Formula(V) or Formula (VI)

Formula (V) and Formula (VI) are Formula (III), where R₁ is OR₃ andNR₄R₅, respectively.

In some embodiments, the prodigiosin analog has the structure of Formula(Va), (Vb), (Vc), (Vd), or (Ve)

Formula (Va) is Formula (V) where R³ is hydrogen. Formula (Vb) isFormula (V) where R³ is benyzl. Formula (Vc) is Formula (V) where R³ isn-butyl. Formula (Vd) is Formula (V) where R₃ is n-octyl. Formula (Ve)is Formula (V) where R³ is 1-pentyne.

In some embodiments, the prodigiosin analog has the structure of Formula(VIa)

Formula (VIa) is Formula (VI), where R⁴ is hydrogen and R⁵ is n-butyl.

In some embodiments, the prodigiosin analog has the structure of Formula(VII)

Formula (VII) is Formula (III) where R₂ is hydrogen.

In some embodiments, the prodigiosin analog has the structure of Formula(VIII)

Formula (VIII) is Formula (III) where R₂ is COR₆.

In some embodiments, the prodigiosin analog has the structure of Formula(IX) or

Formula (X)

Formula (IX) and Formula (X) are Formula (VIII) where R⁶ is OR⁷ orNR⁸R⁹, respectively.

In some embodiments, the prodigiosin analog has the structure of Formula(IXa), Formula (IXb), Formula (IXc), or Formula (IXd)

Formula (IXa) is Formula (IX) where R⁷ is hydrogen. Formula (IXb) isFormula (IX) where R⁷ is ethyl. Formula (IXc) is Formula (IX) where R⁷is n-butyl. Formula (IXd) (PG3-Oc) is Formula (IX) where R⁷ is n-octyl.

In some embodiments, the prodigiosin analog has the structure of Formula(Xa)

Formula (Xa) is Formula (X) where R⁸ is hydrogen and R⁹ is n-butyl.

The compounds may be formulated as a composition, for example, with acarrier. Compositions may comprise a compound of Formulas (I), (II),(III), (IV), (V), (Va), (Vb), (Vc), (Vd), (Ve), (VI), (VIa), (VII),(VIII), (IX), (IXa), (IXb), (IXc), (IXd), (X), (Xa), and (XIV), or apharmaceutically acceptable salt thereof. Compositions may comprise acompound of Formula (XIV), or a pharmaceutically acceptable saltthereof. The composition may include more than one compound, includingany combination, of Formulas (I), (II), (III), (IV), (V), (Va), (Vb),(Vc), (Vd), (Ve), (VI), (VIa), (VII), (VIII), (IX), (IXa), (IXb), (IXc),(IXd), (X), (Xa), and (XIV). The composition may include more than onecompound, including any combination, of compounds within Formula (XIV).The composition may also include one or more other anti-cancer drugs.

The present disclosure also provides compounds of Formula XIV

wherein:

R¹ and R² are, independently, selected from the group consisting of H,OH, halogen, —C₁₋₆alkyl, —C₁₋₆fluoroalkyl, —CN, —NO₂, —SR⁷, —S(═O)R⁷,—S(═O)₂R⁷, —NHS(═O)₂R⁷, —C(═O)R⁷, —OC(═O)R⁷, —CO₂R⁷, —OCO₂R⁷, —CH(R⁷)₂,—N(R⁷)₂, —C(═O)N(R⁷)₂, —NHC(═O)NHR⁷, —NHC(═O)R⁷, —NHC(═O)OR⁷,—C(OH)(R⁷)₂, and —C(NH₂)(R⁷)₂;

each R⁷ is, independently, H, halogen, or C₁-C₆alkyl, wherein the alkylgroup is optionally substituted by 1, 2, 3, 4, or 5 substituentsindependently selected from halogen, OH, CN, and NO₂;

R³ is an optionally substituted aryl or an optionally substitutedheteroaryl;

R⁴, R⁵, and R⁶ are, independently, OH, —C₁₋₁₀alkyl, —OC₁₋₁₀alkyl, or—SC₁₋₁₀alkyl, wherein each alkyl group is, independently, optionallysubstituted by 1, 2, 3, 4, or 5 substituents independently selected fromhalogen, OH, CN, and NO₂; and

n is an integer from 0 to 5; provided that if n is 0, then R³ is not anoptionally substituted pyrrolyl; and if n is 2, then the compound is not

or an isomer, tautomer, or solvate thereof, or a pharmaceuticallyacceptable salt thereof.

In some embodiments, R¹ and R² are, independently, selected from thegroup consisting of H, halogen, —C₁₋₆alkyl, —C₁₋₆fluoroalkyl, —OR⁷,—SR⁷, —S(═O)R⁷, —C(═O)R⁷, —OC(═O)R⁷, —CO₂R⁷, —OCO₂R⁷, —CH(R)₂,—C(═O)N(R⁷)₂, —C(OH)(R⁷)₂, and —C(NH₂)(R⁷)₂. In some embodiments, R¹ andR² are, independently, selected from the group consisting of H, halogen,—C₁₋₆alkyl, —OR⁷, —SR⁷, —S(═O)R⁷, —C(═O)R⁷, —OC(═O)R⁷, —CO₂R⁷, —OCO₂R⁷,—CH(R⁷)₂, and —C(OH)(R⁷)₂. In some embodiments, R¹ and R² are,independently, selected from the group consisting of H, —C₁₋₆alkyl,—OR⁷, —C(═O)R⁷, and OC(═O)R⁷. In some embodiments, R¹ and R² are,independently, selected from the group consisting of H, —C₁₋₆alkyl, and—OR⁷. In some embodiments, R¹ is —OR⁷ and R² is H. In some embodiments,R¹ is —OCH₃ and R² is H.

In some embodiments, R³ is an optionally substituted heteroaryl. In someembodiments, R³ is an optionally substituted heteroaryl selected fromthe group consisting of pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,pyridinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl,thiazolyl, indolyl, pyrryl, oxazolyl, pyrazolyl, isoxazolyl, triazolyl,tetrazolyl, indazolyl, isothiazolyl, purinyl, carbazolyl, isoxazolyl,indolinyl, pyranyl, pyrazolyl, triazolyl, oxadiazolyl, thianthrenyl,indolizinyl, isoindolyl, pyrrolyl, and xanthenyl. In some embodiments,R³ is an optionally substituted heteroaryl selected from the groupconsisting of pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyridinyl,furyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl,pyrazolyl, isoxazolyl, indazolyl, isothiazolyl, purinyl, carbazolyl,isoxazolyl, indolinyl, pyranyl, pyrazolyl, oxadiazolyl, and pyrrolyl. Insome embodiments, R³ is an optionally substituted heteroaryl selectedfrom the group consisting of pyridyl, pyrimidinyl, pyridinyl,imidazolyl, indolyl, pyrryl, purinyl, pyranyl, and pyrrolyl. In someembodiments, R³ is an optionally substituted heteroaryl selected fromthe group consisting of pyridyl, pyrimidinyl, pyridinyl, pyrryl, andpyrrolyl. In some embodiments, R³ is an optionally substituted pyrrolyl.

In some embodiments, R⁴, R⁵, and R⁶ are, independently, OH, —C₁₋₆alkylor —OC₁₋₁₀alkyl, wherein each alkyl group is, independently, optionallysubstituted by 1, 2, or 3 substituents independently selected fromhalogen, OH, CN, and NO₂. In some embodiments, R⁴, R⁵, and R⁶ are,independently, OH, —C₁₋₆alkyl or —OC₁₋₈alkyl, wherein each alkyl groupis, independently, optionally substituted by 1 or 2 substituentsindependently selected from halogen and OH. In some embodiments, R⁴ andR⁶ are, independently, —C₁₋₃alkyl, and R⁵ is OH or —OC₁₋₈alkyl, whereineach alkyl group is, independently, optionally substituted by 1 or 2halogens. In some embodiments, R⁴ and R⁶ are, independently, —C₁₋₃alkyl,and R⁵ is —OC₆₋₈alkyl. In some embodiments, n is 2 or 3. In someembodiments, n is 2.

In some embodiments, R¹ and R² are, independently, selected from thegroup consisting of H, —C₁₋₆alkyl, —C₁₋₆fluoroalkyl, halogen, —OR⁷,—SR⁷, —S(═O)R⁷, —C(═O)R⁷, —OC(═O)R⁷, —CO₂R⁷, —OCO₂R⁷, —CH(R⁷)₂,—C(═O)N(R⁷)₂, —C(OH)(R⁷)₂, and —C(NH₂)(R⁷)₂; each R⁷ is, independently,H, halogen, or C₁-C₆alkyl; R³ is an optionally substituted heteroaryl;R⁴, R⁵, and R⁶ are, independently, OH, —C₁₋₆alkyl or —OC₁₋₁₀alkyl,wherein each alkyl group is, independently, optionally substituted by 1,2, or 3 substituents independently selected from halogen, OH, CN, andNO₂; and n is an integer from 1 to 3.

In some embodiments, R¹ and R² are, independently, selected from thegroup consisting of H, —C₁₋₆alkyl, halogen, —OR⁷, —SR⁷, —S(═O)R⁷,—C(═O)R⁷, —OC(═O)R⁷, —CO₂R⁷, —OCO₂R⁷, —CH(R⁷)₂, and —C(OH)(R⁷)₂; each R⁷is, independently, H, halogen, or C₁-C₃alkyl; R³ is an optionallysubstituted heteroaryl selected from the group consisting of pyridyl,pyrimidinyl, pyrazinyl, pyridazinyl, pyridinyl, furyl, thienyl,imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, pyrazolyl, isoxazolyl,indazolyl, isothiazolyl, purinyl, carbazolyl, isoxazolyl, indolinyl,pyranyl, pyrazolyl, oxadiazolyl, and pyrrolyl; R⁴, R⁵, and R⁶ are,independently, OH, —C₁₋₆alkyl or —OC₁₋₈alkyl, wherein each alkyl groupis, independently, optionally substituted by 1 or 2 substituentsindependently selected from halogen and OH; and n is an integer from 1to 3.

In some embodiments, R¹ and R² are, independently, selected from thegroup consisting of H, —C₁₋₆alkyl, —OR⁷, —C(═O)R⁷, and —OC(═O)R⁷; eachR⁷ is, independently, halogen or C₁-C₃alkyl; R³ is an optionallysubstituted heteroaryl selected from the group consisting of pyridyl,pyrimidinyl, pyrazinyl, pyridazinyl, pyridinyl, furyl, thienyl,imidazolyl, indolyl, pyrryl, purinyl, pyranyl, pyrazolyl, oxadiazolyl,and pyrrolyl; R⁴ and R⁶ are, independently, —C₁₋₃alkyl, and R⁵ is OH or—OC₁₋₈alkyl, wherein each alkyl group is, independently, optionallysubstituted by 1 or 2 halogens; and n is 2 or 3.

In some embodiments, R¹ and R² are, independently, selected from thegroup consisting of H, —C₁₋₆alkyl, and —OR⁷; each R⁷ is, independently,halogen or C₁-C₃alkyl; R³ is an optionally substituted heteroarylselected from the group consisting of pyridyl, pyrimidinyl, pyridinyl,imidazolyl, indolyl, pyrryl, purinyl, pyranyl, and pyrrolyl; R⁴ and R⁶are, independently, —C₁₋₃alkyl; R⁵ is —OC₆₋₈alkyl; and n is 2 or 3.

In some embodiments, R¹ is —OR⁷; R² is H; each R⁷ is, independently,halogen, or C₁-C₃alkyl; R³ is an optionally substituted heteroarylselected from the group consisting of pyridyl, pyrimidinyl, pyridinyl,pyrryl, and pyrrolyl; R⁴ and R⁶ are, independently, —C₁₋₃alkyl; R⁵ is—OC₆₋₈alkyl; and n is 2.

In some embodiments, the compound(s) having Formulas (I), (II), (III),(IV), (V), (Va), (Vb), (Vc), (Vd), (Ve), (VI), (VIa), (VII), (VIII),(IX), (IXa), (IXb), (IXc), (IXd), (X), (Xa), and (XIV) or an isomer,tautomer, or solvate thereof, or a pharmaceutically acceptable saltthereof, are a component of a pharmaceutical composition comprising apharmaceutically acceptable carrier. In some embodiments, thecompound(s) having Formula (XIV) or an isomer, tautomer, or solvatethereof, or a pharmaceutically acceptable salt thereof, are a componentof a pharmaceutical composition comprising a pharmaceutically acceptablecarrier.

In some embodiments, the pharmaceutical composition further comprises ananti-cancer agent. As used herein, the phrase “anti-cancer agent” ismeant to include all forms of treatment of cancer including, but notlimited to, traditional chemotherapy (i.e., chemotherapeutic agents,whether they are administered parenterally or orally), immunotherapeuticagents, small molecule enzyme or kinase inhibitors, intravesicaltherapeutic agents, antibody inhibitors of receptors or kinases,antibody-drug conjugates, and radiation therapy.

Examples of chemotherapeutic agents include, but are not limited to,cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate,phenanthriplatin, picoplatin, satraplatin, methotrexate, vincristine,doxorubicin, tunicamycin, oligomycin, bortezomib, MG132, 5-flurouracil,sorafenib, flavopiridol, gemcitabine, taxol, mercaptopurine,thioguanine, hydroxyurea, cytarabine, mitomycin, cyclophosphamide,ifosfamide, nitrosourea, dacarbazine, procarbizine, an etoposide, acampathecin, bleomycin, idarubicin, daunorubicin, dactinomycin,distamycin A, etidium, netropsin, auristatin, amsacrine, prodigiosin,bortexomib, pibenzimol, tomaymycin, duocarmycin SA, plicamycin,mitoxantrone, asparaginase, vinblastine, vinorelbine, paclitaxel,docetaxel, CPT-11, gleevec, erlotinib, gefitinib, ibrutinib, crizotinib,ceritinib, lapatinib, navitoclax, and regorafenib, or any combinationthereof. In some embodiments, the chemotherapeutic agent is acombination of agents, such as, for example,methotrexate/vincristine/doxorubicin/cisplatin (MVAC) orgemcitabine/cisplatin.

Examples of immunotherapeutic agents include, but are not limited to,OPDIVO® (nivolumab), KEYTRUDA® (pembrolizumab), TECENTRIQ®(atezolizumab), IMFINZI® (durvalab), YERVOY® (ipilumumab), or BAVENCIO®(avelumab), or any combination thereof.

In some embodiments, the ratio of the compound(s) having Formulas (I),(II), (III), (IV), (V), (Va), (Vb), (Vc), (Vd), (Ve), (VI), (VIa),(VII), (VIII), (IX), (IXa), (IXb), (IXc), (IXd), (X), (Xa), and (XIV) tothe anti-cancer agent in the pharmaceutical compositon is from about0.01:1 to about 100:1 w/w. In some embodiments, the ratio of thecompound(s) within Formula (XIV) to the anti-cancer agent in thepharmaceutical compositon is from about 0.01:1 to about 100:1 w/w.

The pharmaceutical compostions described herein can be administered to apatient in need thereof in an oral formulation, an intravenousformulation, a topical formulation, an intraperitoneal formulation, anintrapleural formulation, an intravesical formulation, or an intrathecalformulation. The compositions may be formulated in a suitablecontrolled-release vehicle, with an adjuvant, or as a depot formulation.

Pharmaceutically acceptable salts may be acid or base salts.Non-limiting examples of pharmaceutically acceptable salts includesulfates, methosulfates, methanesulfates, pyrosulfates, bisulfates,sulfites, bisulfites, nitrates, besylates, phosphates,monohydrogenphosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates, propionates,decanoates, caprylates, acrylates, formates, isobutyrates, caproates,heptanoates, propiolates, oxalates, malonates, succinates, suberates,sebacates, fumarates, maleates, dioates, benzoates, chlorobenzoates,methylbenzoates, dinitromenzoates, hydroxybenzoates, methoxybenzoates,phthalates, sulfonates, toluenesulfonates, xylenesulfonates,pheylacetates, phenylpropionates, phenylbutyrates, citrates, lactates,γ-hydroxybutyrates, glycollates, tartrates, methanesulfonates,propanesulfonates, mandelates, and other salts customarily used orotherwise FDA-approved.

In some embodiments, the carrier is a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers include, but are notlimited to, aqueous vehicles such as water, alcohol (e.g., ethanol orglycol), saline solutions, dextrose solutions, and balanced saltsolutions, as well as nonaqueous vehicles such as alcohols and oils,including plant or vegetable-derived oils such as olive oil, cottonseedoil, corn oil, canola oil, sesame oil, and other non-toxic oils. Thecompositions may also comprise one or more pharmaceutically acceptableexcipients.

In some embodiments, the compositions comprise an effective amount ofthe compound such as a compound having Formulas (I), (II), (III), (IV),(V), (Va), (Vb), (Vc), (Vd), (Ve), (VI), (VIa), (VII), (VIII), (IX),(IXa), (IXb), (IXc), (IXd), (X), (Xa), and (XIV), or any combinationthereof. In some embodiments, the compositions comprise an effectiveamount of the compound such as a compound having Formula (XIV), or anycombination of compounds therein.

The compositions may be formulated for administration to a subject inany suitable dosage form. The compositions may be formulated for oral,buccal, nasal, transdermal, parenteral, injectable, intravenous,subcutaneous, intramuscular, rectal, or vaginal administration. Thecompositions may be formulated in a suitable controlled-release vehicle,with an adjuvant, or as a depot formulation.

Preparations for parenteral administration include, but are not limitedto, sterile solutions ready for injection, sterile dry soluble productsready to be combined with a solvent just prior to use, including, butnot limited to, hypodermic tablets, sterile suspensions ready forinjection, sterile dry insoluble products ready to be combined with avehicle just prior to use and sterile emulsions.

Solid dosage forms include, but are not limited to, tablets, pills,powders, bulk powders, capsules, granules, and combinations thereof.Solid dosage forms may be prepared as compressed, chewable lozenges andtablets which may be enteric-coated, sugar coated or film-coated. Soliddosage forms may be hard or encased in soft gelatin, and granules andpowders may be provided in non-effervescent or effervescent form. Soliddosage forms may be prepared for dissolution or suspension in a liquidor semi-liquid vehicle prior to administration. Solid dosage forms maybe prepared for immediate release, controlled release, or anycombination thereof. Controlled release includes, but is not limited to,delayed release, sustained release, timed pulsatile release, andlocation-specific pulsatile release, and combinations thereof.

Liquid dosage forms include, but are not limited to, aqueous solutions,emulsions, suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules. Aqueous solutions include, but are notlimited to, elixirs and syrups. Emulsions may be oil-in water orwater-in-oil emulsions.

Pharmaceutically acceptable excipients utilized in solid dosage formsinclude, but are not limited to, coatings, binders, lubricants,diluents, disintegrating agents, coloring agents, flavoring agents,preservatives, sweeteners, and wetting agents. Enteric-coated tablets,due to their enteric-coating, resist the action of stomach acid anddissolve or disintegrate in the neutral or alkaline intestines. Otherexamples of coatings include, but are not limited to, sugar coatings andpolymer coatings. Sweetening agents are useful in the formation ofchewable tablets and lozenges. Pharmaceutically acceptable excipientsused in liquid dosage forms include, but are not limited to, solvents,suspending agents, dispersing agents, emulsifying agents, surfactants,emollients, coloring agents, flavoring agents, preservatives, andsweeteners.

Suitable examples of binders include, but are not limited to, glucosesolution, acacia mucilage, gelatin solution, sucrose and starch paste.Suitable examples of lubricants include, but are not limited to, talc,starch, magnesium or calcium stearate, lycopodium and stearic acid.Suitable examples of diluents include, but are not limited to, lactose,sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.Suitable examples of disintegrating agents include, but are not limitedto, corn starch, potato starch, bentonite, methylcellulose, agar andcarboxymethylcellulose. Suitable examples of emulsifying agents include,but are not limited to, gelatin, acacia, tragacanth, bentonite, andsurfactants such as polyoxyethylene sorbitan monooleate. Suitableexamples of suspending agents include, but are not limited to, sodiumcarboxymethylcellulose, pectin, tragacanth, veegum and acacia.

Suitable examples of coloring agents include, but are not limited to,any of the approved certified water soluble FD and C dyes, mixturesthereof, and water insoluble FD and D dyes suspended on alumina hydrate.Suitable examples of sweetening agents include, but are not limited to,dextrose, sucrose, fructose, lactose, mannitol and artificial sweeteningagents such as saccharin, aspartame, sucralose, acelsulfame potassium,and other artificial sweeteners. Suitable examples of flavoring agentsinclude, but are not limited to, synthetic flavors and natural flavorsextracted from plants such as fruits and mints, and synthetic blends ofcompounds which produce a pleasant sensation. Suitable examples ofwetting agents include, but are not limited to, propylene glycolmonostearate, sorbitan monooleate, diethylene glycol monolaurate andpolyoxyethylene laural ether. Suitable examples of enteric-coatingsinclude, but are not limited to, fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Suitable examplesof film coatings include, but are not limited to, hydroxyethylcellulose,sodium carboxymethylcellulose, polyethylene glycol 4000 and celluloseacetate phthalate. Suitable examples of preservatives include, but arenot limited to, glycerin, methyl and propylparaben, ethylparaben,butylparaben, isobutylparaben, isopropylparaben, benzylparaben, citrate,benzoic acid, sodium benzoate and alcohol.

Suitable examples of elixirs include, but are not limited to, clear,sweetened, hydroalcoholic preparations. Pharmaceutically acceptablecarriers used in elixirs include solvents. Suiatbel examples of syrupsinclude, but are not limited to, concentrated aqueous solutions of asugar, for example, sucrose, and may contain a preservative. An emulsionis a two-phase system in which one liquid is dispersed throughoutanother liquid. Pharmaceutically acceptable carriers used in emulsionscan also include emulsifying agents and preservatives. Suspensions mayuse pharmaceutically acceptable suspending agents and preservatives.Pharmaceutically acceptable substances used in non-effervescentgranules, to be reconstituted into a liquid oral dosage form, include,but are not limited to, diluents, sweeteners, and wetting agents.Pharmaceutically acceptable substances used in effervescent granules, tobe reconstituted into a liquid oral dosage form, include, but are notlimited to, organic acids and a source of carbon dioxide. Sources ofcarbon dioxide include, but are not limited to, sodium bicarbonate andsodium carbonate. Coloring and flavoring agents may be used in all suchdosage forms.

Additional excipients that may be included in any dosage forms include,but are not limited to, antimicrobial agents, isotonic agents, buffers,antioxidants, local anesthetic agents, sequestering or chelating agents,analgesic agents, antiemetic agents, and other agents to enhanceselected characteristics of the formulation.

Antimicrobial agents may be cidal or static, and may be antimicrobial,antifungal, antiparasitic, or antiviral. Suitable examples of commonlyused antimicrobial agents include, but are not limited to, phenols orcresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Acidic or basic pH may be used for antimicrobialeffects in some aspects. Suitable examples of isotonic agents include,but are not limited to, sodium chloride and dextrose. Suitable examplesof buffers include, but are not limited to, phosphate and citratebuffers. A non-limiting example of a chelating agent for metal ions isEDTA.

The amount of the compound(s) having Formulas (I), (II), (III), (IV),(V), (Va), (Vb), (Vc), (Vd), (Ve), (VI), (VIa), (VII), (VIII), (IX),(IXa), (IXb), (IXc), (IXd), (X), (Xa), and (XIV) to be administered maybe that amount which is therapeutically effective. The dosage to beadministered may depend on the characteristics of the subject beingtreated, e.g., the particular animal treated, age, weight, health, typesof concurrent treatment, if any, and frequency of treatments, and on thenature and extent of the cancer, and can be easily determined by oneskilled in the art (e.g., by the clinician). The selection of thespecific dose regimen can be selected or adjusted or titrated by theclinician according to methods known to the clinician to obtain thedesired clinical response. In addition, in vitro or in vivo assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the compositions may also depend on theroute of administration, and should be decided according to the judgmentof the practitioner and each patient's circumstances.

The compositions may be prepared to provide from about 0.05 mg to about500 mg of the compound of any of the formulas disclosed herein, such asFormula (XIV), or pharmaceutically acceptable salt thereof. In someembodiments, the compositions may comprise from about 1 mg to about 200mg, from about 10 mg to about 200 mg, from about 10 mg to about 100 mg,from about 50 mg to about 100 mg, from about 20 mg to about 400 mg, fromabout 100 mg to about 300 mg, or from about 50 mg to about 250 mg of thecompound of any of the formulas disclosed herein, such as Formula (XIV),or an isomer, tautomer, or solvate thereof, or a pharmaceuticallyacceptable salt thereof.

Suitable dosage ranges for oral administration include, but are notlimited to, from about 0.001 mg/kg body weight to about 200 mg/kg bodyweight, from about 0.01 mg/kg body weight to about 100 mg/kg bodyweight, from about 0.01 mg/kg body weight to about 70 mg/kg body weight,from about 0.1 mg/kg body weight to about 50 mg/kg body weight, from 0.5mg/kg body weight to about 20 mg/kg body weight, or from about 1 mg/kgbody weight to about 10 mg/kg body weight. In some embodiments, the oraldose is about 5 mg/kg body weight.

Suitable dosage ranges for intravenous administration include, but arenot limited to, from about 0.01 mg/kg body weight to about 500 mg/kgbody weight, from about 0.1 mg/kg body weight to about 100 mg/kg bodyweight, from about 1 mg/kg body weight to about 50 mg/kg body weight, orfrom about 10 mg/kg body weight to about 35 mg/kg body weight.

Suitable dosage ranges for other routes of administration can becalculated based on the forgoing dosages as known by one skilled in theart. For example, recommended dosages for intradermal, intramuscular,intraperitoneal, subcutaneous, epidural, sublingual, intracerebral,transdermal, or inhalation are in the range from about 0.001 mg/kg bodyweight to about 200 mg/kg body weight, from about 0.01 mg/kg body weightto about 100 mg/kg body weight, from about 0.1 mg/kg body weight toabout 50 mg/kg body weight, or from about 1 mg/kg body weight to about20 mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.Such animal models and systems are well known in the art.

The disclosure also provides methods for reactivation of the p53pathway. Such methods may comprise treatment methods, by whichreactivation of the p53 pathway treats any condition in which the p53pathway plays a role, including cancer.

In some embodiments, the methods of treatment further compriseadministering another therapy to the subject. In some embodiments, theanother therapy is radiation therapy, chemotherapy, immunotherapy, or acombination thereof. In some embodiments, the another therapy isadministered to the subject at a lower level compared to the level whenadministered in the absence of the compound(s) of Formulas (I), (II),(III), (IV), (V), (Va), (Vb), (Vc), (Vd), (Ve), (VI), (VIa), (VII),(VIII), (IX), (IXa), (IXb), (IXc), (IXd), (X), (Xa), and (XIV). In someembodiments, the another therapy is administered to the subject at alower level compared to the level when administered in the absence ofthe compound(s) of Formula (XIV).

In some embodiments, the methods comprise contacting a cell with mutatedp53 with an effective amount of a compound or composition comprising anyof the formulas described herein, such as Formula (XIV), or anycombination thereof, or any pharmaceutically acceptable salt thereof.The composition may comprise any dosage form and/or any excipients,including those described or exemplified herein.

In some embodiments, the methods comprise contacting a cancer cell withan effective amount of a compound or composition comprising any one ofFormulas (I), (II), (III), (IV), (V), (Va), (Vb), (Vc), (Vd), (Ve),(VI), (VIa), (VII), (VIII), (IX), (IXa), (IXb), (IXc), (IXd), (X), (Xa),and (XIV), or any combination thereof, or any pharmaceuticallyacceptable salt thereof. In some embodiments, the methods comprisecontacting a cancer cell with an effective amount of a compound orcomposition comprising Formula (XIV), or any pharmaceutically acceptablesalt thereof. The composition may comprise any dosage form and/or anyexcipients, including those described or exemplified herein.

In some embodiments, the methods comprise contacting a cell having a p53mutation with an effective amount of a compound or compositioncomprising any one of Formulas (I), (II), (III), (IV), (V), (Va), (Vb),(Vc), (Vd), (Ve), (VI), (VIa), (VII), (VIII), (IX), (IXa), (IXb), (IXc),(IXd), (X), (Xa), and (XIV), or any combination thereof, or anypharmaceutically acceptable salt thereof. In some embodiments, themethods comprise contacting a cell having a p53 mutation with aneffective amount of a compound or composition comprising any one of thecompounds within Formula (XIV), or any pharmaceutically acceptable saltthereof. The composition may comprise any dosage form and/or anyexcipients, including those described or exemplified herein. Incontacting the cell in this manner, the compound or compositionreactivates the p53 pathway. The cell may be within the body of asubject. The cell may be a cancer cell, such as a prostate cancer cell,a breast cancer cell, a kidney cancer cell, an ovarian cancer cell, alymphoma cell, a melanoma cell, a leukemia cell, or a glioblastoma cell.

In some embodiments, methods for treating a cancer patient compriseadministering to the patient a compound or composition comprising anyone of Formulas (I), (II), (III), (IV), (V), (Va), (Vb), (Vc), (Vd),(Ve), (VI), (VIa), (VII), (VIII), (IX), (IXa), (IXb), (IXc), (IXd), (X),(Xa), and (XIV), or any combination thereof, or any pharmaceuticallyacceptable salt thereof, in an amount effective to treat the cancer. Insome embodiments, methods for treating a cancer patient compriseadministering to the patient a compound or composition comprising any ofthe compounds within Formula (XIV), or any pharmaceutically acceptablesalt thereof, in an amount effective to treat the cancer. In someembodiments, the effective amount is an amount effective to reactivatethe p53 pathway in cancer cells within the patient's body. In someembodiments, the patient is a human cancer patient. In some embodiments,the cancer is associated with a p53 gain of function (GOF) mutation. Thecancer may be any cancer in which the p53 pathway is mutated including,but are not limited to, prostate cancer, breast cancer, kidney cancer,ovarian cancer, lymphoma, leukemia, melanoma, or glioblastoma.

In some embodiments, the cancer is selected from the group consisting ofa carcinoma, a sarcoma, a colorectal cancer, a lymphoma, a leukemia, ablastoma, a germ cell cancer, a breast cancer, a lung cancer, apancreatic cancer, a stomach cancer, a bone cancer, an ovarian cancer, aprostate cancer, a head and neck cancer, a bladder cancer, a cervicalcancer, a colon cancer, a skin cancer, a gliobastoma cancer, anesophageal cancer, an oral cancer, a gallbladder cancer, a liver cancer,a testicular cancer, a uterine cancer, a thyroid cancer, and a throatcancer. In some embodiments, the cancer is a colorectal cancer, a headand neck cancer, a pancreatic cancer, a breast cancer, a colon cancer, alung cancer, or a gliobastoma cancer.

Administration may be according to any technique or route suitable tothe cancer being treated or the patient's needs. Administration may be,for example, oral, parenteral, or via direct injection. Administrationmay be directly to the tumor or to a location proximal to the tumor.Delivery may be via the bloodstream. Delivery may include activetargeting, for example, by conjugating the compound to an antibody thatbinds to an antigen on the tumor being treated. Delivery may also bepassive.

Uses of one or more compounds which reactivate the p53 pathway accordingto any one of Formulas (I), (II), (III), (IV), (V), (Va), (Vb), (Vc),(Vd), (Ve), (VI), (VIa), (VII), (VIII), (IX), (IXa), (IXb), (IXc),(IXd), (X), (Xa), and (XIV), or a pharmaceutically acceptable saltthereof, or a composition thereof, in the treatment of cancer or tumorsare also provided. The disclosure provides compounds which reactivatesthe p53 pathway according to any one of Formulas (I), (II), (III), (IV),(V), (Va), (Vb), (Vc), (Vd), (Ve), (VI), (VIa), (VII), (VIII), (IX),(IXa), (IXb), (IXc), (IXd), (X), (Xa), and (XIV), or a pharmaceuticallyacceptable salt thereof, or a composition thereof, in the treatment ofprostate cancer. The disclosure provides compounds which reactivate thep53 pathway according to any one of Formulas (I), (II), (III), (IV),(V), (Va), (Vb), (Vc), (Vd), (Ve), (VI), (VIa), (VII), (VIII), (IX),(IXa), (IXb), (IXc), (IXd), (X), (Xa), and (XIV), or a pharmaceuticallyacceptable salt thereof, or a composition thereof, in the treatment ofkidney cancer. The disclosure provides uses of compounds whichreactivate the p53 pathway according to any one of Formulas (I), (II),(III), (IV), (V), (Va), (Vb), (Vc), (Vd), (Ve), (VI), (VIa), (VII),(VIII), (IX), (IXa), (IXb), (IXc), (IXd), (X), (Xa), and (XIV), or apharmaceutically acceptable salt thereof, or a composition thereof, inthe treatment of breast cancer. The disclosure provides uses ofcompounds which reactivate the p53 pathway according to any one ofFormulas (I), (II), (III), (IV), (V), (Va), (Vb), (Vc), (Vd), (Ve),(VI), (VIa), (VII), (VIII), (IX), (IXa), (IXb), (IXc), (IXd), (X), (Xa),and (XIV), or a pharmaceutically acceptable salt thereof, or acomposition thereof, in the treatment of ovarian cancer. The disclosureprovides uses of compounds which reactivates the p53 pathway accordingto any one of Formulas (I), (II), (III), (IV), (V), (Va), (Vb), (Vc),(Vd), (Ve), (VI), (VIa), (VII), (VIII), (IX), (IXa), (IXb), (IXc),(IXd), (X), (Xa), and (XIV), or a pharmaceutically acceptable saltthereof, or a composition thereof, in the treatment of melanoma. Thedisclosure provides uses of compounds which reactivate the p53 pathwayaccording to any one of Formulas (I), (II), (III), (IV), (V), (Va),(Vb), (Vc), (Vd), (Ve), (VI), (VIa), (VII), (VIII), (IX), (IXa), (IXb),(IXc), (IXd), (X), (Xa), and (XIV), or a pharmaceutically acceptablesalt thereof, or a composition thereof, in the treatment of lymphoma.The disclosure provides uses of compounds which reactivate the p53pathway according to any one of Formulas (I), (II), (III), (IV), (V),(Va), (Vb), (Vc), (Vd), (Ve), (VI), (VIa), (VII), (VIII), (IX), (IXa),(IXb), (IXc), (IXd), (X), (Xa), and (XIV), or a pharmaceuticallyacceptable salt thereof, or a composition thereof, in the treatment ofleukemia. The disclosure provides uses of compounds which reactivate thep53 pathway according to any one of Formulas (I), (II), (III), (IV),(V), (Va), (Vb), (Vc), (Vd), (Ve), (VI), (VIa), (VII), (VIII), (IX),(IXa), (IXb), (IXc), (IXd), (X), (Xa), and (XIV), or a pharmaceuticallyacceptable salt thereof, or a composition thereof, in the treatment ofglioblastoma. Uses may be in the manufacture of a medicament for cancertreatment as provided.

The present disclosure also provides methods of preparing a compound ofFormula XIV

wherein:

R¹ and R² are, independently, selected from the group consisting of H,OH, halogen, —C₁₋₆fluoroalkyl, —CN, —NO₂, —OR⁷, —SR⁷, —S(═O)R⁷,—S(═O)₂R⁷, —NHS(═O)₂R⁷, —C(═O)R⁷, —OC(═O)R⁷, —CO₂R⁷, —OCO₂R⁷, —CH(R⁷)₂,—N(R⁷)₂, —C(═O)N(R⁷)₂, —NHC(═O)NHR⁷, —NHC(═O)R⁷, —NHC(═O)OR⁷,—C(OH)(R⁷)₂, and —C(NH₂)(R⁷)₂;

each R⁷ is, independently, H, halogen, or C₁-C₆alkyl, wherein the alkylgroup is optionally substituted by 1, 2, 3, 4, or 5 substituentsindependently selected from halogen, OH, CN, and NO₂;

R³ is an optionally substituted aryl or an optionally substitutedheteroaryl;

R⁴, R⁵, and R⁶ are, independently, —OH, —C₁₋₁₀alkyl, —OC₁₋₁₀alkyl, or—SC₁₋₁₀alkyl, wherein each alkyl group is, independently, optionallysubstituted by 1, 2, 3, 4, or 5 substituents independently selected fromhalogen, OH, CN, and NO₂; and

n is an integer from 0 to 5;

wherein the method comprises:

admixing a solvent, an acid, a compound of Formula XII

and a compound of Formula XIII

at a temperature sufficient to result in the formation of the compoundof Formula XIV.

In some embodiments, the acid is selected from the group consisting ofhydrochloric acid, hydrobromic acid, hydrofluoric acid, acetic acid,phosphoric acid, toluenesulfonic acid, sulfuric acid, and nitric acid.

In some embodiments, the temperature is about from about 0° C. to about100° C.

In some embodiments, the solvent is a protic solvent. In someembodiments, the solvent is an aprotic solvent.

In some embodiments, the method set forth above further comprisesalkylating the compound of Formula XIII, when R⁵ is OH, prior toreacting with the compound of Formula XII, by admixing the compound ofFormula XIII with an alkylating agent, a base, a nucleophilic catalystsalt, and a solvent at a temperature sufficient to result in theformation of the compound of Formula XIV. In some embodiments, thealkylating agent is selected from the group consisting of a linear—C₁₋₆haloalkyl, a linear —C₁₋₂₀haloalkyl, a branched —C₁₋₆haloalkyl, anda branched —C₁₋₂₀haloalkyl. In some embodiments, the base is selectedfrom the group consisting of potassium carbonate, cesium carbonate,sodium carbonate, and calcium carbonate. In some embodiments, thenucleophilic catalyst salt is selected from the group consisting ofpotassium iodide, sodium iodide, calcium iodide, and tetra-n-butylammonium iodide. In some embodiments, the solvent is a protic solvent.In some embodiments, the solvent is an aprotic solvent. In someembodiments, the temperature is from about 0° C. to about 100° C.

In some embodiments, the method set forth above further comprisesalkylating the compound of Formula XIV, when R⁵ is OH, after reactingthe compound of Formula XIII with the compound of Formula XII, byadmixing the compound of Formula XIV with an alkylating agent, a base, anucleophilic catalyst salt, and a solvent at a temperature sufficient toresult in the formation of the compound of Formula XIV. In someembodiments, the alkylating agent is selected from the group consistingof a linear —C₁₋₆haloalkyl, a linear —C₁₋₂₀haloalkyl, a branched—C₁₋₆haloalkyl, and a branched —C₁₋₂₀haloalkyl. In some embodiments, thebase is selected from the group consisting of potassium carbonate,cesium carbonate, sodium carbonate, and calcium carbonate. In someembodiments, the nucleophilic catalyst salt is selected from the groupconsisting of potassium iodide, sodium iodide, calcium iodide, andtetra-n-butyl ammonium iodide. In some embodiments, the solvent is aprotic solvent. In some embodiments, the solvent is an aprotic solvent.In some embodiments, the temperature is from about 0° C. to about 100°C.

The following examples are provided to further describe the disclosedembodiments in even greater detail. The examples are intended toillustrate, and not to limit, the embodiments disclosed herein.

Example 1: Prodigiosin Analogs and Cell Lines

As used herein, P01 is prodigiosin, P104 is the prodigiosin analog ofFormula (VIa), P105 is the prodigiosin analog of Formula (Vb), P107 isthe prodigiosin analog of Formula (Vd), P108 is the prodigiosin analogof Formula (Ve), P109 is the prodigiosin analog of Formula (Va), P106 isthe prodigiosin analog of Formula (Vc), P301 is the prodigiosin analogof Formula (VII), P302 is the prodigiosin analog of Formula (Xa), P303is the prodigiosin analog of Formula (IXb), P304 is the prodigiosinanalog of Formula (IXa), P305 is the prodigiosin analog of Formula(IXc), P306 is the prodigiosin analog of Formula (IXd), and P01RC isObatoclax. P101 is another prodigiosin analog of Formula (XI).

Various cell lines were obtained for testing the anti-cancer propertiesof the prodigiosin analogs described above. SW480, DLD-1, DLD1-p73KD,HCT116, and p53-null HCT116 were generated in the laboratory and eachcell stably expressed a p-53 regulated luciferase reporter. MRCS andWi38 were obtained from the ATCC and cultured as recommended. Cells wereregularly authenticated by bioluminescence, growth, and morphologicobservation.

p53-Responsive Luciferase Reporter Assay

The p53-mutant SW480 human colon cancer cells, stably expressing ap53-responsive luciferase reporter, were used for compound screening.The SW480 cells were treated with P01, P101, P104, P105, P106, P107,P108, P109, P301, P302, P303, and P304 in concentrations ranging from0.03 μM to 10 μM for 4 hours. After the treatment, cells were imaged byusing an IVIS Imaging System (Xenogen) to detect luciferase activity(see, FIGS. 1, 2A, and 2B). Positive hits with strong activity forluciferase induction were selected for additional testing.

Western Blotting

After treatment, protein lysates were collected for Western blotanalysis. Twenty-five micrograms of protein were used for SDS-PAGE.After primary and secondary antibody incubations, the signal wasdetected with a chemiluminescent detection kit, followed byautoradiography or Syngen. In FIG. 3, SW480 cells were treated with P01,P301, and P303 in various concentrations for 16 hours, and tested for p53, p′73, and Ran proteins. In FIGS. 4-6, DLD-1 and DLD1-p73KD cellswere treated with P01 (see, FIG. 4), P301 (see, FIG. 5), and P303 (see,FIG. 6) in various concentrations for 18 hours, and tested for p21,Noxa, DR5, p′73, and Ran proteins. In FIG. 7, SW480 cells were treatedwith P01 and P306 in various concentrations for 18 hours and tested forp′73, ΔNp73, p53, DR5, Puma, Noxa, P21, and Ran proteins. In FIG. 8,DLD1 and DLD1-p73KD cells were treated with P306 and Cispatlin for 18hours, and tested for p′73, ANp73, p53, DR5, Puma, Noxa, P21, and Ranproteins. In FIG. 9, p53-null HCT116 cells were treated with P01 andP306 in various concentrations for 18 hours, and tested for p′73, ANp73,p53, DR5, Puma, Noxa, P21, and Ran proteins. In FIG. 10, p53-null HCT116cells were treated with P01 and P306, alone or in combination with SiConor SiTAp73, in various concentrations for 6 hours, and tested for p73(C.S.), p73 (Bethyl), P63-α, p53, DR5, Puma, P21, and Ran proteins.

CellTiter-Glo® Luminescent Cell Viability Assay

SW480, DLD-1, DLD1-p73KD, HCT116, and p53-null HCT116, MRCS and Wi38cells were seeded at 5,000 cells per well on 96-well plates. The cellswere treated for 72 hours with P01, P301, P303, P305, P306, and P01RC invarious concentrations. Then, cells were mixed with an equal volume ofCellTiterGlo® reagents (Promega), following the manufacturer's protocol,and bioluminescence imaging was measured using the IVIS imager. Theresults of the luminescent cell viability assay are presented in FIGS.11-16.

Flow Cytometry Assay

After treatment with P01, P301, and P303 in various concentrations for72 hours, SW480 (see, FIG. 17), HT29 (see, FIG. 18), and DLD-1 (see,FIG. 19) cells were harvested, fixed by ethanol, and stained bypropidium iodide. Flow cytometry was then performed on the resultingcells.

Colony Formation Assay

6-well plates were filled with 500 cells per well of SW480 (see, FIG.20) and HT29 (see, FIG. 21) cells. The cells were then treated with P01,P301, and P303 in various concentrations for 72 hours. The cells werethen cultured with drug-free complete medium for 2 weeks with freshmedium changed every 3 days. Cells were fixed with 10% formalin andstained with 0.05% crystal violet at the end of 2 weeks period of cellculture.

Immunofluorescence

SW480 cells were seeded in four-chamber slides. After treatment withP01, P301, P303, and Irinotecan (CPT-11) at various concentrations for 8hours, cells were fixed by Cytofix/Cytoperm (BD Biosciences) for 30minutes. Untreated cells were also fixed as a control. Western blottingwas used to test for γ-H2AX, H3, and Ran proteins in the cells treatedwith P01, P301, P303 (see, FIGS. 22 and 23). Fixed cells were blockedfor 2 hours, followed by primary antibody incubation for 2 hours andsecondary antibody incubation for 2 hours at room temperature. Afterwashing, samples mounted and were examined by fluorescence microscopy(see, FIGS. 24 and 25).

As can be seen from the experimental results, at least prodigiosinanalogs P301 (i.e., Formula (VII)), P303 (i.e., Formula (Xa)), and P306(i.e., Formula (IXd)) potently induced cell death of p53 mutant coloncancer cell line SW480, DLD1 and p53-null cell line HCT116. The IC₅₀values are within nanomolar range. The prodigiosin analogs induced celldeath in cancer cells with no genotoxicity. P301 and P303 induced theexpression of p53-target genes via p73. P306 induced mutant p53 andANp73 degradation and the expression of p53-target genes.

Example 2: PG3-Oc (Formula (IXd)) Materials and Methods

1) Cell lines: HT29, SW480, DLD-1, HCT116, and p53-null HCT116 cells,H1975, MDA-MD-231, U251, FaDu, CAL-27, PANC-1, Aspc-1, and MRCS wereobtained from the ATCC and cultured as recommended. Cells were regularlyauthenticated by bioluminescence, growth, and morphologic observation.The cells were routinely examined for Mycoplasma and all cell linesunderwent STR authentication.

2) Western blotting: After treatment, protein lysates were collected forWestern blot analysis. 15 μg of protein was used for SDS-PAGE. Afterprimary and secondary antibody incubations, the signal was detected bychemiluminescent detection kit, imaged by Syngene (Imgen Technologies).Antibodies for Puma, FLIP_(L/S) and p53 (Santa Cruz Biotechnology),cleaved caspase 8, caspase 9, caspase 3, cleavage PARP, eIF2α,p-eIF2α(Ser51), CHOP, ATF4, DR5, FOXO3a, p-FOXO3a(Ser253), NF-κB p65,p-NF-κB p65(Ser536), c-Jun, p-c-Jun(Ser63), JNK, p-JNK(Thr183/Tyr185)(Cell Signaling Technology), Noxa, p21 (Calbiochem), p73 (Bethyllaboratories Inc), Ran (BD Biosciences), β-actin (Sigma).

3) Cell viability assay: Cells were seeded in 96-well plate (6×10³cells/well). Cells were treated with different concentrations ofcompounds or dimethyl sulfoxide (DMSO) control for 72 hours. The cellviability was assessed by CellTiterGlo bioluminescent cell proliferationassay (Promega), following the manufacturer's protocol. Bioluminescenceimaging was measured using the IVIS imager. Percentage of cell viability(mean±SEM) at each dose was calculated against the respective DMSOcontrol. The IC₅₀ values were determined from the sigmoidaldose-response curves using GraphPad Prims4.

4) Caspase activity assay: Cells were seeded in 96-well plate (1×10⁴cells/well). Cells were treated with different concentrations ofcompounds or DMSO control for 24 hours. The caspase 3/7 activity wasassessed by Caspase-Glo® 3/7 Assay kit (Promega), following themanufacturer's protocol. Bioluminescence imaging was measured using theIVIS imager. Caspase activity was normalized to cell numbers andcompared to those of DMSO treatment as control in each cell line. Datais reported as mean RLU+SEM (n=3).

5) Colony formation assays: Five hundred cells were seeded per well on6-well plates and treated with different concentrations of compounds for24 hours, then, cells were cultured with drug-free complete medium for 2weeks with fresh medium changed every 7 days. Cells were fixed with 10%formalin and stained with 0.05% crystal violet at the end of 2 weeksperiod of cell culture.

6) Flow cytometry assay:

a) Cell Cycle Analysis: Propidium iodide (PI) staining and flowcytometry were used to determine the degree of cellular apoptosis. Cellswere seeded at 3×10⁵ cells/well in six-well plates. Cells were treatedwith PG3-Oc for 48 hours. Cells were harvested, fixed by 70% ethanol,and stained by propidium iodide, then flow cytometry was performed aspreviously described (Smithen et al., Org. Biomol. Chem., 2013, 11,62-68). The percentage of hypodiploid cells (sub-G1) was used toquantify dead cells in apoptosis assays.

b) Early apoptosis detection: Cells were seeded at 3×10⁵ cells/well insix-well plates. Cells were treated with PG3-Oc for 48 hours. Cells wereharvested and prepared using Alex Fluor 488 Annexin V/Dead CellApoptosis Kit following manufacturer's protocol (Thermo ScientificInvitrogen).

7) Real-time reverse transcriptase PCR: Total RNA was isolated fromPG3-Oc-treated cells using Qick-RNA mini prep kit (Zymo Research,Irvine, Calif.) according to the manufacturer's protocol. 500 ng oftotal RNA was used to generate cDNA using SuperScript III first-strandsynthesis system with random primers (Invitrogen), following themanufacturer's protocol. Real-time PCR was performed using POWER SYBRGREEN mast mix (Applied Biosystem) for DR5, p21, PUMA and GAPDH on7900HT Sequence Detection System (Applied Biosystem). PUMA primer(forward, 5′-GACGACCTCAACGCACAGTA-3′ (SEQ ID NO:1); reverse,5′-AGGAGTCCCATGATGAGATTGT-3′ (SEQ ID NO:2)), DR5 primer (forward,5′-ACAGTTGCAGCCGTAGTCTTG-3′ (SEQ ID NO:3); reverse, 5′-CCAGGTCGTTGTGAGCTTCT-3′ (SEQ ID NO:4)), GAPDH primer (forward,5′-TCGACAGTCAGCCGCATCTTCTTT-3′ (SEQ ID NO:5); reverse,5′-ACCAAATCCGTTGACTCCGACCTT-3′ (SEQ ID NO:6)). ΔΔCt method was used toanalyze and report fold change of indicated genes.

8) siRNA knockdown: Knockdown experiments were performed by transfectingeither 80 pmole of indicated siRNA(s), or scramble siRNA using RNAiMAX(Invitrogen). Transfected cells were treated with PG3-Oc, 24 hourspost-transfection. The control scrambled siRNA and siRNA for human ATF4,CHOP, DR5, Puma, NF-κB p65 were purchased from Santa Cruz Biotechnology.p73 siRNA was from Ambion, and FOXO3a siRNA from Thermo ScientificDharmacon.

9) Knock-out of PUMA by CRISPR/Cas9 gene editing:

a) sgRNA design and plasmid construction: sgRNA targets the exon 3 ofPUMA gene, which contains sequence code for BH3 domain of PUMA. TwosgDNAs (Guide 1 and Guide 2) were introduced into lentiviral vectors(pLentiCRISPR-E) which contain eSpCas9 and puromycin cassette. GuidelDNA (forward, 5′-CACCGGCGGGCGGTCCCACCCAGG-3′ (SEQ ID NO:7); reverse,5′-AAACCCTGGGTGGGACCGCCCGCC-3′ (SEQ ID NO:8)) and Guide 2 DNA (forward,5′-CACCGCCGCTCGTACTGTGCGTTG-3′ (SEQ ID NO:9); reverse,5′-AAACCAACGCACAGTACGAGCGGC-3′ (SEQ ID NO:10)) were annealed and linkedto the restriction enzyme-cut plasmid by T4 ligase. Stb13 strain(Invitrogen C7373-03) was transformed by the guides-containing plasmids.LB-amp plates were streaked and incubated on a shaker at 37 C overnight.The bacteria colonies were selected and mixed up with LB (TerrificBroth) and 100 μg/mL ampicillin, and were incubated on a shaker at 37 Covernight. Plasmids from different colonies were isolated and purifiedusing QIAprep Spin Miniprep Kit (Qiagen). To screen plucks, plasmidswere digested with EcoR I HF and Bam HI in Cut Smart Buffer (New EnglandBioLabs, Inc.) at 37 C for 1 hour and then analyzed by 1% agarose gel.Sequencing was performed by GENEWIZ (South Plainfield, N.J.; see, FIGS.30A-30E and FIGS. 34A-34I).

b) Cell culture, DNA transfection: Lentivirus were generated withpsPAX2, pVSV-G and the pLentiCRISPR plasmids that contain the guides andCas9 in 293T cells. 48 hours later, all the supernatant was transferredto a 1.5 mL tube. The debris was removed by centrifugation and thesupernatant was ransferred to a new 1.5 mL tube, and stored at 4 C. HT29cells were transfected with the lentivirus supernatant and polybrene wasadded to enhance the transfection. Puromycin at a final concentration of1 μg/mL was added to medium to select positive cells.

c) Mutation screens by Sanger sequencing and TIDE analysis: DNA wasextracted and purified from positive HT29 cells using DNeasy Blood &Tissue kit (Qiagen). PCR primers that flank both sides of the exon 3 ofPUMA gene were used to amplify the target region (forward,5′-CACAGTCTCTGGCCTTCTGG-3′ (SEQ ID NO:11); reverse, 5′-AGCTGCCGCACATCTGG-3′ (SEQ ID NO:12)). The amplicon is GC-rich region, to improve PCRspecificity. Temperature gradient PCR was performed to optimizeannealing temperature. A hot-start and touch-down PCR with accuPrime™Pfx DNA Polymerase (ThermoFisher Scientific) and 2.5% DMSO and 1Mbetaine, was performed to achieve specific amplification of targetregion. The PCR products were purified by QIAquick PCR purification kit(Qiagen) for Sanger sequencing.

TIDE analysis was performed using an online tool (TIDE: Tracking ofIndels by Decomposition (see, world wide web at“tide-calculator.nki.nl/”)). Sequencing was performed by GENEWIZ (SouthPlainfield, N.J.; see, FIGS. 30A-30E and FIGS. 34A-34I).

d) Single cell colonies. 300 positive HT29 cells were placed into a 10cm dish and incubated at 37 C. After 2 weeks, single cell colonies wereselected and expanded. Western blotting using PUMA antibody wasperformed to screen the colonies (see, FIGS. 30A-30E and FIGS. 34A-34I).

10) Statistical analysis: All results were obtained from triplicateexperiments, unless other indicated. Statistical analyses were performedusing PRISM4 Software (GraphPad Software, Inc.), and the Student t test.Statistical significances were determined by P<0.05. Combination indiceswere calculated using the Chou-Talalay method with CalcuSyn software(Biosoft).

Results

1) PG3-Oc Inhibits Growth in a Broad Panel of p53-Mutant Cancer CellLines:

Efficacy of the newly synthesized analogs was assayed by measuring cellviability, at 72 hours post-treatment. Of the 15 compounds screened,PG3-Oc (see, FIG. 26A) was identified as the most potent inhibitor ofcell growth in a broad spectrum of human cancer cells with mutant p53.These included colorectal cancer cell lines (HT29, SW480, DLD1, HCT116and HCT116 p53^(−/−)) and head and neck squamous cell lines (FaDu andCAL-27) (see, FIGS. 26B, 26C and 26D). IC₅₀ values for pancreatic cancercell lines (PANC-1 and ASPC-1), glioblastoma (U251), non-small cell lungcancer (H1975) and triple-negative breast cancer cells (MDA-MB-231 andMDA-MB-468) were within the nano-molar range (see, FIG. 26B). Thepotency of PG3-Oc (Oc) for inhibition of cancer cell growth was found tobe comparable with prodigiosin (P) and obatoclax (Ob) (see, FIG. 26D).PG3-Oc showed similar toxicity for normal cell MRCS as obatoclax (see,FIG. 26E). For colorectal and head and neck squamous cancer cells, theIC₅₀ for normal cells was found to be about 3-fold higher than thevalues in colorectal and head-neck cancer cells. These data indicatethat PG3-Oc can be a suitable compound in the treatment of humancolorectal cancers.

In particular, referring to FIGS. 26A-26E, PG3-Oc inhibition of thegrowth of p53-mutant cancer cell lines is shown. FIG. 26A shows thestructure of PG3-Oc. FIG. 26B shows dose response curves and EC₅₀ valuesof PG3-Oc in a panel of cancer cell lines with p53 mutation, comparingto normal human cells MRCS. FIG. 26C shows colony formation assay ofp53-mutant and p53-null human cancer cells. Cells were treated withindicated concentrations of PG3-Oc for 24 hours, and then cultured indrug-free medium for 14 days following crystal violet staining ofattached cells. FIG. 26D shows cell viability assay, comparing potencyof PG3-Oc (Oc) to obatoclax (Ob) and prodigiosin (P) in p53 wild typecell line HCT116 and p53 mutant cell line SW480. Cells were treated withdifferent concentration of PG3-Oc or DMSO control for 72 hours.Luciferase activity was imaged by the IVIS Imaging System aftertreatment. Cell viability data were normalized to those of DMSOtreatment as control in each cell line and data analyses were performedusing PRISM4 software. EC₅₀ data are expressed as mean±SD in normalfibroblast cells (normal; n=3). FIG. 26E shows cell viability assay,comparing toxicity of PG3-Oc (Oc) to obatoclax (Ob) in MRCS cells.

2) PG3-Oc Induces Apoptosis in Mutant p53-Expressing Human Cancer CellLines:

Treatment of colorectal cancer cell lines HT29 and SW480 with 1 μMPG3-Oc for 48 hours induced cancer cell death as demonstrated by sub-G1analysis (see, FIG. 27A). To evaluate if the cell death wascaspase-dependent, Caspase 3/7 activity was measured. Treatment withPG3-Oc induced a 2-fold increase in caspase 3/7 activity as compared tountreated cells using mutant p53 and p53-null expressing cancer cells(see, FIG. 27B). Induction of apoptosis was further confirmed bypan-caspase inhibitor (Z-VAD-FMK) co-treatment experiments with PG3-Oc.As seen in FIG. 27C, 20 μM Z-VAD-FMK completely blocked the formation ofa sub-G1 population as compared to the untreated control. Under similarexperiment conditions, western blot analysis showed that Z-VAD-FMK (20μM) completely inhibits the cleavage of caspase-8 and caspase-3 in bothHT29 and SW480 cells (see, FIGS. 27D and 27E). Taken together, thesedata indicate that PG3-Oc treatment induces capase-8 and caspase-3activation in colorectal cancer cell lines, and caspase activation maybe required for PG3-Oc-induced cell death.

In particular, referring to FIGS. 27A-27E, PG3-Oc-induced apoptosis inp53 mutant cancer cell lines is shown. FIG. 27A shows cell-cycleprofiles of cells at 48 hours after PG3-Oc treatment. Apoptosis wasanalyzed by nuclear PI-staining using flow cytometry. HT29 and SW480cells were treated with PG3-Oc at indicated concentration for 48 hours,DLD1 cells were treated for 72 hours. FIG. 27B shows caspase 3/7activity assay. Cells were treated with PG3-Oc at the indicatedconcentrations for 24 hours. Luciferase activity was imaged by the IVISImaging System after treatment. Caspases activity data (triplicate) werenormalized to cell numbers and then those of DMSO treatment as controlin each cell line and data analyses were performed using Excel. FIG. 27Cshows HT29 cells were co-treated with 1 μM PG3-Oc and pan-caspaseinhibitor Z-VAD-fmk for 48 hours. Cell cycle analysis was performed asbefore. Western blotting analysis of active caspase-8, active caspase-3and cleaved PARP in HT29 cells (see, FIG. 27D) and SW480 cells (see,FIG. 27E).

3) PG3-Oc Restores p53 Pathway in p53 Mutant Cancer Cell Lines:

Similar to prodigision, treatment of p53 mutant containing SW480 andp53-null HCT116 colon cancer cells with PG3-Oc also potently inducedup-regulation of p53 target genes, such as DR5, PUMA, Noxa and p21 (see,FIGS. 28A and 28B). However, the magnitude of induction of target geneswas much higher in PG3-Oc treated cells as compared to prodigiosin,especially for p21 and PUMA (see, FIGS. 28A and 28B). To investigatewhether the up-regulation of p53 target genes occurs at thetranscriptional level, after cells were treated with 1 μM PG3-Oc atdifferent time points, real-time PCR analysis of mRNA level of DR5, p21and PUMA was performed in HT29 and HCT116 p53^(−/−) cells (see, FIGS.28C and 28D). At 8 and 19 hour time points, robust up-regulation of bothp21 and PUMA mRNAs were observed in the cell lines tested. For DR5 mRNAlevel, more than 2-fold up-regulation was observed at 19 hourspost-treatment in HT29 cells. Contrary to that, DR5 protein level waspotently up-regulated in HCT116 p53^(−/−) cells with no significantchange of DR5 mRNA. This indicates that PG3-Oc treatment may lead to DR5protein stabilization depending on cell type. Taken together, these dataindicate that PG3-Oc can restore the p53 pathway at the transcriptionallevel, especially for p21 and PUMA.

In particular, referring to FIGS. 28A-28D, PG3-Oc restoration of the p53pathway in p53 mutant cancer cell lines is shown. FIGS. 28A and 28B showPG3-Oc induced expression of p53-target genes in p53-mutant cell lines.PG3-Oc induced more up-regulation of Puma and p21 than prodigiosin (P)in both p53-muant SW480 (see, FIG. 28A) and p53-null HCT 116 cancer celllines (see, FIG. 28B). Western blot analysis of p53-target geneexpression of DR5, Puma, Noxa and p21 in p53-mutat and p53-null cancercells. Cells were treated with PG3-Oc at indicated concentrations for 18hours. FIGS. 28C and 28D show qPCR analysis of the change of mRNA levelin HT29 and HCT116 p53−/−. Cells were treated with PG3-Oc (1 μM) for 8hours and 19 hours. mRNA samples were prepared and RT-PCR was performedto prepare cDNAs.

4) PUMA is Required for PG3-Oc Mediated Cell Death:

Whether PUMA and DR5 are dispensable for PG3-Oc mediated cell death inmutant p53 cells was examined. Since PUMA was most dramatically inducedby PG3-Oc in HT29 cells, this cell line was selected to dissect out therole of PUMA. Time-course experiments indicated that PUMA protein wasfirst induced at 16 hours post PG3-Oc treatment and this induction wassustained even at 48 hours. At 48 hours, induction of cleaved PARP wasobserved, as well as cleaved caspase-8 and -3 occurred (see, FIG. 29B).Therefore, 48 hours as a time period was selected for a subsequentdose-response study of PG3-Oc (see, FIGS. 29A and 29C). These dataindicate that PG3-Oc induces up-regulation of PUMA in a time-and-dosedependent manner. A similar time- and dose-dependent induction of DR5was observed in PG3-Oc treated cells.

Having optimized the time and dose of PG3-Oc using different apoptosismarkers, siRNA studies were subsequently performed. As shown in FIGS.29D and 29E, knockdown of PUMA by siRNA reduced the sub-G1 population to11.1% as compared to 25.8% in siControl, in PG3-Oc treated cells.However, knockdown of DR5 by siRNA did not protect cells from deathinduced by PG3-Oc (see, FIG. 29D). Similar results were observed byWestern Blot analysis when PUMA was knocked down alone or together withDR5 using siRNA. As shown in FIG. 29E, PUMA knockdown completely bluntedPARP cleavage and cleavage of caspases post PG3-Oc treatment. However,DR5 knockdown had no impact on the same apoptotic markers. Takentogether, this indicates that DR5 is dispensable for PG3-Oc mediatedcell death. However, PUMA protein is required and is a key player incell death induced by PG3-Oc treatment in HT29 cancer cells.

In particular, referring to FIGS. 29A-29E, the correlation of inductionof PUMA with cell death is shown. FIGS. 29A, 29B, and 29C showdose-response and time-course analysis of active caspase-3, activecaspase-8, active caspase-9, cleaved PARP (cPARP), Puma, and DR5 inPG3-Oc-treated HT29 cells (see, FIGS. 29A and 29B) or SW480 cells (see,FIG. 29C) by Western Blot. FIG. 29D shows HT29 cells transfected withControl, Puma, DR5 and Puma/DR5 siRNAs, after 24 hours transfection, thecells were treated with 1 μM PG3-Oc for 48 hours. After treatment,apoptosis was analyzed by nuclear PI-staining using flow cytometry. FIG.29E shows Western blotting analysis of Puma, DR5, active caspase-8,caspase-9 caspase-3 and cleaved PARP.

PUMA siRNA studies were validated by creating PUMA gene knockout HT29cells line via CRISPR/Cas9 gene editing technology (see, FIGS. 30A-30E).The guide was designed to target the DNA sequence that encodesamino-acid residues for the BH3-domain of PUMA (see, FIG. 30A). Knockoutof the PUMA gene was found to abolish PG3-Oc-induced sub-G1 population,as well as cleavage of PARP and caspases (see, FIGS. 30F and 30G). Thisfurther indicates that binding of PUMA to anti-apoptotic Bcl-2 familymembers (Bcl-2, Mcl-1) may be important for PG3-Oc-mediated cell death.This may be due to disruption of the BH3-domain of PUMA and abrogationof the downstream mediators of apoptosis.

Usually activation of caspase-8 involves the extrinsic pathway ofapoptosis. Of note, both knockout of the PUMA gene and knockdown of PUMAmRNAs not only abolished caspase-8 cleavage induced by PG3-Oc treatment,but also inhibited the cleavage of caspase-9, caspase-3 and PARP (see,FIGS. 29E and 30G). Further, blockage of caspase-8 by the caspase-8inhibitor Z-IETD-FMK not only inhibited caspase 8 cleavage, but alsoresulted in inhibition of cleavage of caspase-9, caspase-3 and PARP. Inaddition, the caspase-8 inhibitor completely blocked the sub-G1population induced by PG3-Oc treatment (see, FIGS. 30F and 30G). Bycontrast, the caspase-9 inhibitor Z-LEHD-FMK partially abrogatedPG3-Oc-induced activation of caspase 3 and cleavage of PARP (see, FIGS.30F and 30G). Combined treatment of caspase-8 and 9 inhibitors preventedcleavage of both caspase-3 and PARP, and reduced the sub-G1 populationto the same level as untreated control cells. The pan-caspase inhibitorZ-VAD-FMK inhibited the formation of a sub-G1 population and blocked thecleavage of caspase-8, caspase-3, caspase-9 and PARP, similar toknockout of PUMA or knockdown of PUMA (see, FIGS. 30F and 30G).

In particular, referring to FIGS. 30A-30H, it is shown that PUMA is akey effector of PG3-Oc mediated apoptosis in mutant p53 cell lines. FIG.30A shows the human PUMA gene contains three coding exons (exons-2-4)and two non-coding exons (exons 1a and 1b). PUMA protein has twofunctional domains, the BH3 and C-terminal mitochondria-localizaionsignal (MLS). The red-colored residues are conserved within otherproapoptotic Bcl-2 family members. FIG. 30B shows sequencing result ofguide 1-containing plasmid P1D. FIG. 30C shows DNA sequencing results ofHT29-P1D, which are pools of lentivirus-infected and puromycin-selectedcells. FIG. 30D shows the decomposition window of TIDE analysis forHT29-P1D. FIG. 30E shows Western blotting analysis of the express ofPUMA protein from single cell colonies of HT29-P1D cells. FIG. 30F showsHT29 and HT29-Puma-KO cells were treated with PG3-Oc or co-treated withcaspase 8(cas8 inh), caspase-9(cas9 inh) and pan-caspase (Z-VAD-FMK)inhibitors for 48 hours, subG1 populations were analyzed by flowcytometry. FIG. 30G shows the activation of caspases and PARP cleavagewere detected by western blotting using indicated antibodies. FIG. 30Hshows a model for PUMA mediated activation of caspase-8.

Taken together, these data indicate that caspase-8 cleavage is anup-stream event of the activation of caspase-9 and caspase-3, and thatPUMA mediates the apoptotic effects of PG3-Oc through activationcaspase-8.

5) The Molecular Mechanism of PG3-Oc-Induced Up-Regulation of PUMA MayInvolve the UPR:

The molecular mechanisms responsible for up-regulation of p53 targetgenes by PG3-Oc in p53 mutant colorectal cancer cells was investigated.Transcription factors p73, p63, ATF4, CHOP, FOXO3a, NF-κB, and JNK/c-Juncan mediate induction of PUMA in a p53-independent manner depending oncell types and stimuli.

PG3-Oc treatment resulted in a decrease of p73 protein in DLD1, HCT116p53^(−/−) (see, FIGS. 31A and 31C), SW480 and HT29 (FIGS. 32A and 32B).Knockdown of p73 did not affect PG3-Oc-induced up-regulation of p53target genes, DR5, p21, Noxa and PUMA (see, FIGS. 31A and 31C). Thesedata indicate that p73 is not involved in PG3-Oc-induced up-regulationof these p53 target genes in these colorectal cancer cell lines.

In particular, referring to FIGS. 32A and 32B, the time-course analysisof active caspase-3, active caspase-8, active caspase-9, cleavedPARP(cPARP), Puma, and DR5 in PG3-Oc-treated HT29 cells (see, FIG. 32A)or HCT116 p53^(−/−) cells (see, FIG. 32B) by Western Blot. Regorafenib(Rego) is a positive control for Puma, and thapsigargin(Tg) is apositive control for DR5.

PG3-Oc treatment resulted in up-regulation of ATF4 and CHOP in both DLD1and HCT116 p53^(−/−) cell lines. However, induction of ATF4 and CHOPoccurred at a significantly lower concentration in HCT116 p53^(−/−)cells at 1.25 μM as compared to 5 μM in DLD1 cells (see, FIGS. 33C and33D). HCT116 p53^(−/−) cells were selected for studying whether ATF4and/or CHOP may be responsible for PUMA up-regulation. Knockdown of ATF4or CHOP by siRNAs, respectively, did not blunt up-regulation of PUMA andp21, but blocked the up-regulation of DR5. These data indicate that ATF4and CHOP are not involved in regulation of PUMA and p21, but may beresponsible for DR5 induction (see, FIG. 31B), indicating that PG3-Octreatment may trigger the UPR signaling pathway.

PG3-Oc treatment leads to decreased phosphorylation of Ser-253 ofFOXO3a, increased phosphorylation of Ser-536 of NF-κB p56 andphosphorylation of JNK and c-Jun (see, FIGS. 33A and 33B); however,knockdown of FOXO3a and NF-κB p56, inhibition of JNK by JNK inhibitorSP600125 did not abolish up-regulation of PUMA (see, FIGS. 31D and 31E).These data indicate that NF-κB, FOXO3a and JNK/c-Jun do not involved inthe regulation of Puma.

In particular, referring to FIGS. 33A-33D, the exploration of themolecular mechanism of PG3-Oc-induced up-regulation of PUMA is shown.HT29 cells were treated with indicated doses and time points,phosphorylation of FOXO3a, NF-κB, JNK, c-Jun and Erk1/2 were detected byWeston blotting using corresponding antibodies.

In particular, referring to FIGS. 31A-31E, the exploration of themolecular mechanism of PG3-Oc-induced up-regulation of PUMA is shown.FIG. 31A shows p53 mutant DLD1(S241F) and p73 stable-knockdownDLD1-p73KD were treated with indicated concentration of PG3-Oc for 18hours, Cisplatin was used as a positive control for p73. FIG. 31B showsHCT116 p53^(−/−) cells were transfected with ATF4 or CHOP siRNAs, after24 hours transfection the cells were treated with PG3-Oc for 24 hours.Protein levels of p53 target genes in cells were detected by WesternBlot. FIG. 31C shows HCT116 p53^(−/−) cells were transfected with p73siRNA, after 24 hours transfection the cells were treated with PG3-Ocfor 6 hours. Protein levels of p53 target genes in cells after PG3-Octreatment were detected by Western Blot. Knockdown of p73 does notprevent PG3-Oc-induced expression of p53-target genes. FIG. 31D showsHT29 cells were transfected with Control, NF-κB and FOXO3a siRNAsrespectively. After 24 hours of transfection, the cells were treatedwith 1 μM PG3-Oc for 24 hours and protein levels in cells were detectedby Western Blot. FIG. 31E shows T29 cells were pre-treated for 1 hourwith JNK inhibitor SP600125, and then treated with 1 μM PG3-Oc for 24hours. Protein levels in cells were detected by western blot usingindicated antibodies.

In particular, referring to FIGS. 34A-34I, the knock-out of PUMA byCRISPR/Cas9 gene editing is shown. FIG. 34A shows P1A, P1D, P2A and P2Bare plasmids containing guide 1 or guide 2 purified from correspondingbacteria colonies (P1A, P1D, P2A and P2B), respectively. Plcve is anegative control plasmid, and KDM6A is a positive control plasmid. FIG.34B shows sequence of guide 2 and sequencing result of guide2-containing plasmid P2A. FIG. 34C shows PCR results of HT29-P1D,HT29-P2A and SW780 (wild-type DNA) using primers that cover the exon 3of PUMA gene. FIG. 34D shows DNA sequencing results of HT29-P2A, whichis a pool of lentivirus-infected and puromycin-selected cells. FIG. 34Eshows the decomposition window and indel spectrum of TIDE analysis forHT29-P2A. FIG. 34F shows indel spectrum of TIDE analysis for HT29-P2A.FIG. 34G shows indel spectrum of TIDE analysis for HT29-P1D. FIG. 34Hshows Western blotting analysis of the express of PUMA protein inHT29-P1D and HT29-P2A cells. FIG. 34I shows Western blotting analysis ofthe express of PUMA protein from single cell colonies of HT29-P1D cells.

Discussion:

Apoptosis repressor with caspase recruitment domain (ARC) is anendogenous inhibitor of apoptosis which binds and suppresses caspase-8.Expression of ARC protein is predominantly seen in terminallydifferentiated cells (cardiac, skeletal myocytes and neurons) undernormal conditions and is markedly induced in a variety of cancersincluding pancreatic, colorectal, breast, lung, glioblastoma, liver,kidney, melanoma, and acute myeloid leukemia. ARC is a primary target ofp53, and p53 transcriptionally represses the express of ARC, which caninitiate apoptosis. Phosphorylation of ARC at T149 by CK2 (casein kinase2) leads to ARC translocation from cytosol to mitochondria where itbinds to death domain of caspase-8 and inhibits caspase-8 activation.

PUMA localizes in mitochondria and induces apoptosis by activatingcaspases via activating BAK and BAX to cause mitochondrial dysfunction.ARC binds to caspase-8 death domain through its N-terminal CARD (caspaserecruitment domain) domain. PUMA via its BH3 domain binds to the CARDdomain of ARC tightly, resulting in releasing of caspase-8 from ARC, andthen activation of caspase-8. Vice versa, up-regulation of ARC proteinlevel in cancer cells can suppress PUMA-mediated caspase activation andapoptosis by sequestering PUMA and releasing anti-apoptotic Bcl-2 familymembers. Based on the data, a model of PG3-Oc-induced and PUMA-mediatedapoptosis in colorectal cancer cells is disclosed in FIG. 30H.

The results indicate that a prodigiosin analog, PG3-Oc, has comparableefficacy as obatoclax and prodigiosin in p53 mutant cancer cell lines.PG3-Oc is a more potent inducer than prodigiosin in restoration of thep53 signaling pathway.

Example 3: Synthesis of PG3-Oc (Formula (IXd))

A representative synthesis of PG3-Oc and related compounds is shown inFIGS. 35 and 39. Compound 1 and Compound 2 (see, FIG. 39) were purchasedfrom AstaTech Inc. (Bristal, Pa. 19007).

Synthesis of compound 4 (PG3-0c): Mass spectrum analysis was performedwith Waters LC-MS system which includes a Waters single quadrupole 3100MS (mass detector using electrospray and chemical ionization). ¹H NMRanalysis was performed on a Bruker Advance 300 MHz instrument (see,FIGS. 36, 37, and 38 MS and NMR spectrums).

Compound 3,(Z)-3-(5-((4′-methoxy-1H,5′H-[2,2′-bipyrrol]-5′-ylidene)methyl)-2,4-dimethyl-1H-pyrrol-3-yl)propanoicacid (Compound 3)

2,4-Dimethyl-1H-pyrrole-3-carboxylic acid 131.9 mg (compound 1, 0.79mmol) and 4-methoxy-1H,1′H-2,2′-bipyrrole-5-carbaldehyde 100 mg(compound 2, 0.53 mmol) were dissolved in 10 mL ethanol, and then 90 μLconcentrated hydrochloric acid was added to the mixture. The reactionwas stirred at room temperature for 3 hours. The reaction mixture wasconcentrated. The crude material was chromatographed 63-200 μM aluminumoxide (activity II) eluting with ethyl acetate/hexane 30:70 to producethe desired compound 3, giving a correct molecular weight 339.91. ¹H NMR(300 MHz, DMSO-d6): δ=11.4 (1H, bs), 7.66 (1H, s), 7.59 (1H, s), 7.21(1H, s), 6.95 (1H, s), 4.22 (3H, s), 2.83 (2H, t), 2.64 (3H, s), 2.55(2H, t), 2.38 (3H, s).

Compound 4 (PG3-Oc), Octyl(Z)-3-(5-((4′-methoxy-1H,5′H-[2,2′-bipyrrol]-5′-ylidene)methyl)-2,4-dimethyl-1H-pyrrol-3-yl)propanoate

KI (18.7 mg), Cs₂CO₃ (169.3 mg) and compound 3 (75 mg) were added to0.75 ml anhydrous DMF, stirred for 5 minutes at room temperature. Then1-Bromooctane (39 μL) was added to the mixture, which was stirred atroom temperature for 24 hours. 20 ml of PBS w/o Ca²⁺—Mg²⁺ buffer wasadded to the reaction mixture. The mixture was extracted with 20 mL×2dichloromethane, and combined organic layer was washed with 50 ml ofsaturated NaCl. The organic layer was dried over anhydrous Na₂SO₄overnight. The next day, the dried organic layer was concentrated andcrude product was separated on aluminum oxide column. The desiredcompound 4 was eluted with ethyl acetate/hexane gradient from 10% to20%. MS analysis gave the correct molecular weight [M+H⁺] 452.23. ¹H NMR(300 MHz, CDCl₃): δ=6.88 (1H, s), 6.63 (2H, s), 6.12 (1H, s), 6.06 (1H,s), 4.00 (2H, s), 3.98 (3H, s), 2.57 (2H, t), 2.31 (2H, s), 2.12 (3H,s), 1.76 (3H, s), 1.55 (3H, m), 1.26 (10H, m), 0.87 (3H, t).

Example 4: Materials and Methods of Cell-Based Biological Evaluation ofPG3-Oc Cell Lines

P53-mutant cell lines: HT29 (R273H), SW480 (R273H/P309S), DLD-1 (S241F),H1975 (R273H), MDA-MD-231 (R280K), U251 (R273H), FaDu (R248L), CAL-27(H193L), PANC-1 (R273H), Aspc-1 (frameshift mutation), Jurkat (multiplep53 mutations, including truncation); P53 wild-type cell lines: HCT116,and CCD 841 Con; P53-null cell line: HCT116 p53^(−/−). All cell lineswere obtained from the ATCC and cultured as recommended. Cells wereregularly authenticated by bioluminescence, growth, and morphologicobservation. Cells were routinely checked for mycoplasma and all celllines underwent STR authentication.

Western Blotting

After treatment, protein lysates were collected for Western blotanalysis. A total of 15 μg of protein was used for SDS-PAGE. Afterprimary and secondary antibody incubations, the signal was detected by achemiluminescence detection kit, imaged by Syngene (Imgen Technologies).Antibodies for PUMA (for IHC), p53 (Santa Cruz Biotechnology), caspase8, cleaved caspase 8, caspase 9, caspase 3, cleavage PARP, eIF2a,p-eIF2a (Ser51), CHOP, ATF4, DR5, FOXO3a, p-FOXO3a (Ser253), NF-κB p65,p-NF-κB p65 (Ser536), c-Jun, p-c-Jun (Ser63), JNK, p-JNK(Thr183/Tyr185), PUMA (for WB), c-Myc, phosphor-S62-cMyc (Cell SignalingTechnology), Noxa, p21 (Calbiochem), p73 (Bethyl laboratories Inc), Ran(BD Biosciences), β-actin (Sigma).

Cell Viability Assay

Cells were seeded in 96-well plates (6×10³ cells/well). Cells weretreated with different concentrations of compounds or dimethyl sulfoxide(DMSO) as a control for 72 hours.

The cell viability was assessed by CellTiterGlo bioluminescent cellproliferation assay (Promega), following the manufacturer's protocol.Bioluminescence imaging was measured using the IVIS imager. Percentageof cell viability (mean±SEM) at each dose was calculated against therespective DMSO control. The IC50 values were determined from thesigmoidal dose-response curves using GraphPad Prism.

Caspase Activity Assay

Cells were seeded in 96-well plate (1×10⁴ cells/well). Cells weretreated with different concentrations of compounds or dimethyl sulfoxide(DMSO) as a control for 24 hours. Caspase 3/7 activity was assessed bythe Caspase-Glo® 3/7 Assay kit (Promega), following the manufacturer'sprotocol. Bioluminescence imaging was measured using the IVIS imager.Caspase activity was normalized to cell numbers and compared to those ofthe DMSO treatment control in each cell line. Data is reported as meanRLU+SEM (n=3).

Colony Formation Assays

Five hundred cells were seeded per well in 6-well plates and treatedwith different concentrations of compounds for 24 hours, then, cellswere cultured with drug-free complete medium for 2 weeks with freshmedium changed every 7 days. Cells were fixed with 10% formalin andstained with 0.05% crystal violet at the end of 2 weeks period of cellculture (Franken et al., Nat. Protoc., 2006, 1, 2315-9).

Cell Uptake and Localization

A total of 5×10⁴ cells were seeded in each well of 8-well chamberslides. Cells were incubated with PG3-Oc for 2 and 8 hours respectively,washed and fixed by 4% paraformaldehyde for 15 minutes at roomtemperature, washed, stained with DAPI for 10 minutes, mounted, andexamined by fluorescence microscopy.

Immunofluorescence Staining

A total of 5×10⁴ cells were seeded in each well of 8-well chamberslides. After treatment, cells were fixed and permeabilized bymethanol:acetone (1:1) for 20 minutes at −20° C. Fixed cells wereblocked by 2% BSA for 1 hour, followed by primary antibody incubationfor 1 hours and Cy3-conjuated secondary antibody incubation for 1 hourat room temperature. After washing, cells were stained with DAPI for 10minutes at room temperature. Cells were mounted, and examined byfluorescence microscopy.

Flow Cytometry Assay

Cell Cycle Analysis: Propidium iodide (PI) staining and flow cytometrywere used to determine the degree of cellular apoptosis. Cells wereseeded at 3×10⁵ cells/well in six-well plates. Cells were treated withPG3-Oc for 48 hours. Cells were harvested, fixed by 70% ethanol, andstained by propidium iodide, then flow cytometry was performed aspreviously described (Smithen et al., Org. Biomol. Chem., 2013, 11,62-68). The percentage of hypo-diploid cells (sub-G1) was used toquantify dead cells in apoptosis assays.

qRT-PCR

Total RNA was isolated from PG3-Oc-treated cells using the Quick-RNAmini prep kit (Zymo Research, Irvine, Calif.) according to themanufacturer's protocol. 500 ng of total RNA was used to generate cDNAusing SuperScript III first-strand synthesis system with random primers(Invitrogen), following the manufacturer's protocol. Real-time PCR wasperformed using POWER SYBR GREEN mast mix (Applied Biosystem) for DR5,p21, PUMA, GAPDH, and TaqMan primer-probes for detection of c-Myc mRNAlevels on 7900HT Sequence Detection System (Applied Biosystem). Primershaving SEQ ID NOs:1-6 (see above) were used. Taq Prob IDs for c-Myc (HS00153408) and GAPDH (HA 99999905). ΔΔCt method was used to analyze andreport fold changes of the indicated genes.

siRNA Knockdown

Knockdown experiments were performed by transfecting either 80 pmoles ofindicated siRNA(s), or scramble siRNA using RNAiMAX (Invitrogen).Transfected cells were treated with PG3-Oc, 24 hours post-transfection.The control scrambled siRNA and siRNA for human ATF4, CHOP, DR5, Puma,NF-κB p65, and c-Myc were purchased from Santa Cruz Biotechnology. p73siRNA was obtained from Ambion, and FOXO3a siRNA was obtained fromThermo Scientific Dharmacon.

Transfection of Plasmids

Cells were transfected with c-Myc expression plasmids (Ricci et al.,Mol. Cell. Biol., 2004, 24, 8541-55) and vector pcDNA3 (Invitrogen)using Lipofectamine 2000 (Invitrogen) according to the manufacturer'sinstruction.

Immunoprecipitation of PUMA with ARC

After 48 hours of co-transfection of PUMA and ARC plasmids usingLipofectamine 2000, HEK 293 cells were lysed with immunoprecipitationlysis buffer. 300 μg whole-cell lysate were incubated with 5 μg ARCantibody for 6 hours at 4° C. and followed by adding 10 μL of proteinA/G Sepharose beads, and the samples were rocked at 4° C. for overnightand then washed three times with 200 μL washing buffer. Samples wereeluted with elution buffer, followed by SDS-PAGE to detect ARC and PUMA.

Knock-Out of PUMA by CRISPR/Cas9 Gene Editing

sgRNA design and plasmid construction: sgRNA targets the exon 3 of PUMAgene, which contains sequence code for BH3 domain of PUMA. Two sgDNAs(Guide) was introduced into lentiviral vectors (pLentiCRISPR-E) whichcontain eSpCas9 and puromycin cassette. Guidel DNA primers (SEQ IDNO:7-8) and Guide 2 DNA primers (SEQ ID NO:9-10) (see above) wereannealed and linked to the restriction enzyme-cut plasmid by T4 ligase.Stb13 strain (Invitrogen C7373-03) was transformed by theguides-containing plasmids. LB-amp plates were streaked and incubated ona shaker at 37 C overnight. The bacterial colonies were selected andmixed with LB (Terrific Broth) and 100 μg/mL of ampicillin, and wereincubated on a shaker at 37 C overnight. Plasmids from differentcolonies were isolated and purified using QIAprep Spin Miniprep Kit(Qiagen). Plasmids were digested with EcoRI HF and BamHI in Cut SmartBuffer (New England BioLabs, Inc.) at 37 C for 1 hour and then analyzedby 1% agarose gel. Sequencing was performed by GENEWIZ (SouthPlainfield, N.J.; see FIGS. 11 A-F).

Cell Culture, DNA Transfection

Lentivirus was generated with psPAX2, pVSV-G and the pLentiCRISPRplasmids that contain the guides and Cas9 in 293T cells. 48 hours later,all supernatant was transferred to a 1.5 mL tube. Debris was removed bycentifugation, and the supernatant was transferred to a new 1.5 mL tube,and stored at 4 C. HT29 cells were transfected with the lentivirussupernatant and polybrene was added to enhance the transfection.Puromycin (final concentration is 1 μg/mL) was added to medium to selectpositive cells.

Mutation Screens by Sanger Sequencing and TIDE Analysis

DNA was extracted and purified from positive HT29 cells using DNeasyBlood & Tissue kit (Qiagen). PCR primers that flank both sides of theexon 3 of PUMA gene were used to amplify the target region (usingprimers with SEQ ID NO:1112; see above). The amplicon was GC-rich. Thus,to improve PCR specificity, temperature gradient PCR was performed tooptimize annealing temperature. A hot-start and touch-down PCR withaccuPrime™ Pfx DNA Polymerase (ThermoFisher Scientific) and 2.5% DMSOand 1M betaine, was performed to achieve specific amplification oftarget region. The PCR products were purified by QIAquick PCRpurification kit (Qiagen) for Sanger sequencing. TIDE analysis wasperformed using an online tool (TIDE: Tracking of Indels byDEcomposition, world wide web at “tide-calculator.nki.nl/”). Sequencingwas performed by GENEWIZ (South Plainfield, N.J.; see, FIG. 30C).

Single Cell Colonies

300 positive HT29 cells were placed into a 10 cm dish and incubated at37 C. After 2 weeks, single cell colonies were selected and expanded.Western blotting using PUMA antibody was performed to screen thecolonies (see, FIGS. 95 and 96).

In Vivo Anti-Tumor Assay

One million HT29 were implanted subcutaneously in the flanks in eachathymic nude mouse (female, 4-6 weeks old). The mice were divided atrandom into two groups and treated with the vehicle (10% DMSO, 20%Kollipher EL in PBS) and PG3-Oc (5 mg/kg, 3 times/week) byintraperitoneal injection when the tumor masses reached a size of 5 to 6mm. Subsequently, tumor volumes were measured with a caliper andcalculated using V=0.5×Length×Width. Twenty three days after treatment,the mice were euthanized and tumors were excised. H & E staining andImmunohistochemistry (IHC) of paraffin-embedded tumor and tissuesections were performed at the Fox Chase Cancer Center HistopathologyFacility.

Statistical Analysis

All results were obtained from triplicate experiments, unless otherindicated. Statistical analyses were performed using PRISM4 Software(GraphPad Software, Inc.), and the Student t test. Statisticalsignificances were determined by P<0.05. Combination indices werecalculated using the Chou-Talalay method with CalcuSyn software(Biosoft).

Example 5: PG3-Oc Inhibits Cell Proliferation and Induces Apoptosis inMutant p53-Expressing Cancer Cell Lines

PG3-Oc was a potent inhibitor of cell proliferation, and its potency wasfound to be comparable to prodigiosin and obatoclax (FIGS. 39 and 26D).PG3-Oc was efficacious in a broad spectrum of human cancer cells withmutant p53. IC₅₀ values were within the nano-molar range (Table 1).PG3-Oc had a 4- to 9-fold therapeutic index in colorectal cancer (CRC)cell lines as compared to normal colon cells CCD 841 Con (FIG. 40 andTable 1). In addition, PG3-Oc had anti-proliferative effects on othertumor types, including head and neck squamous cancer cell lines,pancreatic cancer, breast cancer, glioblastoma multiforme and non-smallcell lung cancer cells (NSCLC) (FIG. 41 and Table 1). Like CRC, the IC₅₀in additional tumor types was also in the sub-micromolar range (Table1). Over 90% inhibition in long-term cell proliferation was alsoobserved in a panel of CRC cell lines treated with low dose PG3-Oc(FIGS. 84 and 85). These data suggest that PG3-Oc is a promising leadcompound for further evaluation of efficacy in the treatment of humancolorectal cancers and other solid tumors.

It was determined that PG3-Oc induced cell death in mutantp53-expressing cell lines. Treatment of colorectal cancer cell linesHT29 and SW480 with PG3-Oc induced cancer cell death in dose-and-timedependent manner demonstrated by sub-G1 analysis (FIG. 42, and FIGS. 86and 87).

To evaluate if the cell death was caspase-dependent, apoptosis markerswere analyzed by western blot. As seen in FIG. 29B, as low as 0.5 μMPG3-Oc was sufficient to activate cleaved caspase-8 and -3 andcleaved-PARP. Time-course experiments indicated that PUMA protein wasfirst induced at 16 hours post PG3-Oc treatment and this induction wassustained even at 48 hours. At 48 hours, it was noted that induction ofcleaved PARP, as well as cleaved caspase-8 and -3 occurred in both HT29and SW480 cells (FIGS. 29A and 29C). These data also clearly indicatedthat PG3-Oc induces upregulation of PUMA and DR5 in a dose- andtime-dependent manner.

Caspase-dependent induction of apoptosis was further confirmed by thepan-caspase inhibitor (Z-VAD-FMK) co-treatment experiments with PG3-Oc.As seen in FIG. 27C, 20 μM Z-VAD-FMK completely blocked the formation ofa sub-G1 population as compared to the untreated control. Under the sameexperimental conditions, western blot analysis showed that Z-VAD-FMKcompletely inhibited the cleavage of caspase-8 and caspase-3 in bothHT29 (FIG. 27D) and SW480 cells (FIG. 27E). Caspase 3/7 activity wasalso measured. Treatment with PG3-Oc induced a 2-fold increase incaspase 3/7 activity as compared to untreated cells using mutantp53-expressing and p53-null cancer cells (FIG. 27B). PG3-Oc's apoptoticactivity was found to be p73-independent as evident by the comparablecaspase 3/7 activity in both DLD-1 and DLD1-p73^(−/−) cells post PG3-Octreatment (FIG. 27B). Taken together, these data suggest that PG3-Octreatment induces capase-8 and caspase-3 activation in colorectal cancercell lines, and caspase activation is needed for PG3-Oc-induced celldeath.

TABLE 1 IC₅₀ values for different cancer cell lines with various mutantp53 status Tumor/Tissue P53 Type Cell Line IC₅₀ (nM) status ColorectalCancer HT29 66.3 R273H SW480 95.3 (4-fold) R273, P309S DLD1 54 S241FHCT116 p53^(−/−) 41.1 (9 fold) Null Non-transformed CCD 841 Con 375.2 WTcolorectal epithelial cells Head & neck FaDu 66 R248L squamous cellCAL-27 33.9 H193L carcinoma Pancreatic Cancer PANC-1 135.5 R273H ASPC-139.2 Frameshift Breast Cancer MDA-MB-231 242.3 R280K MDA-MB-468 97.6R273H Glioblastoma U251 100.2 R273H Multiforme NSCLC H1975 190.4 R273H

Example 6: PG3-Oc Restores p53 Pathway Signaling without GenotoxicEffects

Having confirmed that PG3-Oc induces apoptosis in multiplep53-expressing mutant cancer cell lines, whether this small moleculerestored p53 pathway signaling was investigated in HT29 cells aftertreatment with 1 μM PG3-Oc for 24 hours. For this purpose, geneprofiling was performed by RNA-Seq and bioinformatics analysis, such asIPA (Ingenuity pathway analysis), GSEA (gene set enrichment analysis)and GO (gene ontology) (See details at Methods and Materials). IPAanalysis of 1867 altered genes revealed that among of 284 known p53target genes (Fischer, Oncogene, 2017, 36(28), 3943-3956), 35 genes wereup-regulated and 24 genes were down-regulated (FIG. 49). Key p53 targetgene CDKN1A (p21) that negatively regulate cell cycle and other p53target genes that regulate apoptosis such as, TNFRSF10B (DR5), BBC3(PUMA), BIRCS (Survivin), TP53INP1 (Teap) were identified in canonicalp53 pathway analysis. Pro-apoptotic p21, DR5, PUMA and Teap werepotently induced and anti-apoptotic Survivin was significantlydownregulated (FIG. 88). p53 target gene PMAIP1 (Noxa) underwent a1.9-fold increase as compared to control, however the cutoff for thebioinformatics analysis was 2, hence, it was not shown in the IPAanalysis. To verify that the behavior of regulation of p53 target genesby PG3-Oc is similar to p53 protein, based on published data (Fischer,Oncogene, 2017, 36(28), 3943-3956), genes directly repressed (FIG. 89)and activated (FIG. 90) by the p53 transcription factor were selected.It was observed that PG3-Oc treatment potently inhibited gene expressionof p53 negatively-regulated genes besides SCD, and strongly upregulatedgene expression of p53 positively-regulated genes besides HSPA4L, DDB2,and PIDD1 (FIGS. 89 and 90). This implicates that PG3-Oc is able torestore the p53 pathway. This observation was further supported by GSEAanalysis which indicated that PG3-Oc-induced differential expression ofgenes was enriched in the p53 pathway, suggesting PG3-Oc has asignificant impact to restore the p53 pathway and network (FIG. 44).

HT29 cells were treated with 1 μM PG3-Oc at different time pointsfollowed by qRT-PCR analysis. Time-dependent induction of DR5, p21 andPUMA transcripts was observed (FIG. 28C). This is consistent with theRNA-Seq data (FIGS. 88 and 90). Importantly, PG3-Oc very stronglyinduced upregulation of PUMA mRNA in all three cell lines at the 8- or19-hour time points (FIGS. 28C, 28D and 45). Over 3-fold induction ofp21 mRNA was observed at 8 and 19 hours post-treatment in HT29 andHCT116 p53^(−/−) cells, but no significant change was observed in SW480cells. Of note, p21 protein level was potently upregulated in SW480(FIG. 28A). For the DR5 mRNA level, about 2-fold upregulation at 19hours post-treatment was observed in HT29 and SW480 cells, but not inHCT116 p53^(−/−) cells (FIGS. 28C, 28D and 45). Interestingly, DR5protein level was potently upregulated in HCT116 p53^(−/−) cells (FIG.28B). These data suggest PG3-Oc treatment may lead to p21 or DR5 proteinstabilization depending on the cell type.

Western blot analysis of p53 mutant DLD1, SW480, HT29 cells, andp53-null HCT116 colon cancer cells showed strong upregulation of DR5,p21, PUMA and Noxa in a time- and dose-dependent manner (FIGS. 29B, 29Aand 29C; FIGS. 31A, 28A and 28B). Further, the magnitude of induction ofp53 target genes was higher in PG3-Oc treated SW480 and HCT116 p53^(−/−)cells as compared to prodigiosin, especially for PUMA and p21 (FIGS. 28Aand 28B).

It was observed that PG3-Oc treatment led to downregulation of p73 bothat the protein level (FIG. 31A and FIG. 61) and the mRNA level (FIGS. 88and 89). Consistent with this, induced upregulation of DR5, p21, PUMAand Noxa showed no significant differences compared to the p73stable-knockdown DLD1 cell line (FIG. 31A). In addition, caspase3/7activity assay also showed no significant difference between DLD1 andDLD1-p73^(−/−) cell lines (FIG. 27B). Thus, the upregulation of the p53pathway by PG3-Oc was independent of p73. Taken together, these datasuggest that PG3-Oc can restore the p53 pathway in mutant p53-expressingcancer cell lines at the transcriptional level, with especially highinduction observed for PUMA.

DNA damage induces the p53 pathway and leads to cell apoptosis. To studywhether the p53 pathway restoration by compound PG3-Oc is due to DNAdamage, the uptake and localization of PG3-Oc was investigated in cells.PG3-Oc and prodigiosin are red fluorescent compounds, and theirlocalization in live cells can be monitored by fluorescence microscopy.PG3-Oc and prodigiosin rapidly entered cells within 2 hours ofincubation and remained in the cytosol at the 8-hour time point in HT29and SW480 cells (FIG. 46). Since it was already observed that 1 μMPG3-Oc treatment for 8 hours can prominently induce the upregulation ofPUMA mRNA (FIGS. 28C, 28D and 45) in HT29, SW480 and HCT116 p53^(−/−)cells, DNA damage marker γ-H2AX (phospho Ser 139-histone H2AX)expression was investigated after the treatment for 8 hours. Westernblot analysis showed that PG3-Oc and prodigiosin did not induce γ-H2AXin HT29 and SW480 cells at lower doses required for p53 pathwayactivation (FIG. 47). Immunofluorescence staining showed that 1 μMPG3-Oc and prodigiosin did not induce γ-H2AX foci formation after the8-hour treatment. By comparison, DNA damage chemotherapeutic drug CPT-11was used as a positive control, and significantly induced γ-H2AX foci inHT29 and SW480 cells (FIG. 47). Both western blot and immunofluorescencestaining data were consistent with the cytoplasmic localization ofPG3-Oc. A previous publication also indicated that 1 μM prodigiosin didnot induce γ-H2AX in SW480 cells and HCT116 p53^(−/−) cells (Hong etal., Cancer Res, 2014, 74, 1153-1165). These results are also consistentwith other group's results. Baldino, et al. reported that prodigiosinlocalized to the cytoplasm, but not within the nucleus, and no γ-H2AXsignal was detected in A549 cancer cells (Baldino et al., Bioorg MedChem Lett, 2006, 16, 701-704). One paper reported opposite results(Montaner et al., Toxicol Sc, 2005, 85, 870-879). However, thoseexperiments were conducted under very different conditions. For example,MCF7 cells were pre-treated with Cupric acetate (2 μM) and PARPinhibitor DPQ (30 μM) for 30 minutes, and then treated with prodigiosin(2 μM) for 3 hours (Montaner et al., Toxicol Sc, 2005, 85, 870-879).This set of conditions probably is not physiologically relevant. Thedata presented herein indicate that the restoration of the p53 pathwayby PG3-Oc at low concentrations did not show genotoxic effects in mutantp53-expressing cancer cells.

Example 7: PUMA is Required for PG3-Oc Mediated Cell Death, andPG3-Oc-Induced Upregulation of DR5 is Through ATF4/CHOP Axis

Whether PUMA and DR5 are dispensable for PG3-Oc mediated cell death inmutant p53-expressing cells was evaluated. As shown in FIG. 49, whenPUMA was knocked down, alone or together with DR5, using siRNA, therewas complete blunting of PARP cleavage and cleavage of caspases afterPG3-Oc treatment. However, DR5 knockdown alone had no impact on the sameapoptotic markers under the experimental conditions. Similar resultswere seen when knockdown of PUMA by siRNA reduced the sub-G1 populationto 11.1% as compared to 25.8% in siControl, in PG3-Oc treated cells.However, knockdown of DR5 by siRNA did not protect cells from deathinduced by PG3-Oc (FIGS. 49 and 50 and FIG. 29D).

PUMA siRNA studies were validated by creating PUMA gene knockout HT29cells via CRISPR/Cas9 gene-editing technology (FIGS. 30A-30D and 95-96)(For details see Materials and Methods). The gRNA was designed to targetthe DNA sequence that encodes amino-acid residues for the BH3-domain ofPUMA (FIG. 30A). Knockout of the PUMA gene was found to abolishPG3-Oc-induced cleavage of PARP and caspase-8, -3 and sub-G1 populationwere the same as the positive control caspase-8 inhibitor Z-IETD-fmk andthe pan-caspase inhibitor Z-VAD (FIGS. 51 and 52 and FIG. 91). Takentogether, these data suggest that DR5 is dispensable for PG3-Oc mediatedcell death. However, PUMA protein is required and is a key player incell death induced by PG3-Oc treatment in HT29 cancer cells.

Of note, both knockdown and knockout of PUMA gene abolished caspase-8and caspase-3 cleavage/activation and PARP cleavage after PG3-Octreatment (FIGS. 49 and 51). Further, the caspase-8 inhibitor Z-IETD-fmknot only inhibited caspase-8 cleavage, but also resulted in inhibitionof caspase-3 and PARP cleavage. These data suggest that the induced PUMAis able to feedback to mediate the activation of caspase-8 through anunknown mechanism.

PG3-Oc-induced upregulation of DR5 was of interest in terms of mechanismand function. First of all, Western blotting data indicated the PG3-Octreatment potently induced upregulation of ATF4 and CHOP, but the levelof phosph-Ser-eIF2a did not increase compared to untreated controls(FIG. 33D and FIG. 33C), suggesting that PG3-Oc induced upregulation ofATF4 and CHOP might not go through ER stress or the integrated stressresponse pathway. siRNA knockdown of ATF4 led to potent blockage of CHOPand DR5 induction, but not PUMA. Knockdown of CHOP blocked DR5upregulation, but not ATF4 and PUMA (FIG. 53). Taken together, thesedata indicated that PG3-Oc-induced upregulation of DR5 is throughATF4/CHOP axis, and ATF4 or CHOP did not mediate the induction of PUMA.

HT29 is a TRAIL-resistant cell line. Cells were pre-treated with 1 μMPG3-Oc to allow DR5 induction, and then TRAIL was added to the medium atdifferent doses for an additional 5 hours. Cleaved capase-8, -9 and -3were dramatically increased in a dose response manner compared withTRAIL treatment alone (FIG. 54). siRNA knockdown of DR5 potently reducedthe sub-G1 populations of co-treatment with PG3-Oc and TRAIL from 72.9%to 29.5% (FIG. 55). Corresponding western blot data showed thatknockdown of DR5 reduced the level of co-treatment-induced caspase-8cleavage to the level of PG3-Oc treatment alone. Knockdown of caspase-8is a positive control in both experiments (FIGS. 55 and 56). These dataindicate that DR5 upregulation is required to sensitize HT29 cells toTRAIL treatment.

Knockout of the PUMA gene blunted cleavage of caspsase-8, -9, -3 andPARP induced by PG3-Oc and TRAIL co-treatment (FIG. 57). Depletion ofboth DR5 and PUMA further reduced caspase-8 activation induced by PG3-Ocand TRAIL co-treatment, as compared to knockdown of DR5 alone (FIG. 58).Taken together, these results indicated PUMA-mediated caspase-8activation also contributes to sensitization to TRAIL treatment. Insummary, a model as shown in FIG. 59 is proposed.

Example 8: PG3-Oc Dependent Repression of c-Myc Upregulates PUMA

Transcription factors p′73, p63, ATF4, CHOP, FOXO3a, NF-κB, andJNK/c-Jun can regulate PUMA gene expression in a p53-independent mannerdepending on cell types and stimuli (Zhang et al., Cancer Res, 2015, 75,3842-3852; Hong et al., Cancer Res, 2014, 74, 1153-1165; Prabhu et al.,Cancer Res, 2016, 76, 1989-1999; Sun et al., Oncogene, 28, 2348-2357;Dudgeon et al., Mol Cancer Ther, 2010, 9, 2893-2902; Qing et al., CancerCell, 2012, 22, 631-644; Cazanave et al., Am J Physiol GastrointestLiver Physiol, 2010, 299, G236-G243; Ghosh et al., PLos ONE, 2012, 7,e39586; Dudgeon et al., Oncogene, 2012, 31, 4848-4858; Zhao et al.,Biochem J, 2012, 444, 291-301; Zhang et al., Oncogene, 2014, 33,1548-1557; Chen et al., Clin Cancer Res, 2014, 20, 3472-3484; Gao etal., Cell Death Differ, 2010, 17, 699-709). In addition, c-Myc is knownto repress PUMA gene expression (Amente et al., Nucleic Acids Res, 2011,39, 9498-9507 and Yun et al., Blood, 2016, 127, 2711-2722). A candidateapproach was taken and checked which of these factors mediates PUMAupregulation in PG3-Oc treated cells.

Stable knockdown of p73 or siRNA knockdown of p73 and/or p63 did notattenuate PG3-Oc-induced upregulation of PUMA in either DLD1 or HT29cells (FIG. 31A and FIG. 93). These are consistent with the observationof PG3-Oc-treatment resulting in downregulation of p73 protein levels inDLD1 and HT29 cells (FIG. 31A, FIG. 61, FIG. 88 and FIG. 93), and alsowith PG3-Oc-induced caspase3/7 activity showing no significantdifference between DLD1 and DLD1p73^(−/−) cells (FIG. 27B). Knockdown oftranscription factors FOXO3a and NF-κB (p65) respectively, or inhibitionof JNK/c-Jun signaling by JNK inhibitor SP600125 did not bluntPG3-Oc-induced upregulation of PUMA (FIGS. 31D and 31E). Knockdown ofATF4 or CHOP also did not attenuate upregulation of PUMA by PG3-Oc (FIG.53). These data suggest that p73, p63, ATF4, CHOP, NF-κB, FOXO3a, andJNK/c-Jun are not involved in the regulation of PUMA in PG3-Oc treatedcells.

PG3-Oc-induced significant downregulation of c-Myc and upregulation ofPUMA protein levels was observed in a panel of p53 mutant cell lines,such as HT29, DLD1, FaDu, MDA-MB-231, MDA-MB-468, SW480 and CAL27 (FIG.60). Experiments in isogenic HCT116 cells with wild-type p53 or p53-nullshowed no significant differences in induction of PUMA or downregulationof c-Myc by PG3-Oc (FIG. 63). These data suggest that PG3-Oc-induceddownregulation of c-Myc and upregulation of PUMA is not limited to aspecific cell line or p53 mutation, and is independent of p53 status.

Of the different candidates tested, it was found that PG3-Oc treatmentpotently downregulated c-Myc in colorectal cancer cell lines (FIGS. 61and 63) and c-Myc addicted acute T cell leukemia Jurkat cells that carrymultiple p53 mutations (FIG. 62). Interestingly, c-Myc downregulationand PUMA upregulation simultaneously occurred upon treatment withPG3-Oc, suggesting c-Myc might negatively regulate PUMA expression.Basal PUMA levels were modestly de-repressed on knockdown of c-Myc, bothat the protein and mRNA levels (FIGS. 64 and 65). Over-expression ofc-Myc led to attenuation of PUMA induction at both the protein and mRNAlevels (FIGS. 66 and 67) post PG3-Oc treatment. To study whetherendogenous c-Myc can inhibit PG3-Oc-induced upregulation of PUMA or not,HCT116 p53^(−/−) and HT29 cells were co-treated with PG3-Oc andproteasome inhibitor MG132. MG132 blocked c-Myc degradation and led toaccumulation of endogenous c-Myc that abolished PG3-Oc-inducedupregulation of PUMA (FIG. 69).

qPCR data indicated that PG3-Oc treatment did not significantly changec-Myc mRNA levels in either HT29 or HCT116 p53^(−/−) cell lines (FIG.68). Proteasome inhibitor MG132 was able to rescue PG3-Oc-induceddegradation of c-Myc protein in both HT29 and HCT116 p53^(−/−) cells(FIG. 69). Taken together, these data indicate that degradation of c-Mycis through proteasome pathway in PG3-Oc treated tumor cells.

A subset of c-Myc target genes was selected (Fernandez et al., GenesDev, 2003, 17, 1115-1129), and their expression was altered based onRNA-Seq data analysis in PG3-Oc treated cells (FIG. 70). c-Mycpositively regulates TGFB2 (TGFβ-2), HSPE1 (Hsp10), MYBL2 (B-Myb), E2F1(E2F1) and FOXM1 (FOXM1) (Fernandez et al., Genes Dev, 2003, 17,1115-1129). Downregulation of these genes is consistent withPG3-Oc-induced degradation and inhibition of c-Myc (FIGS. 61 and 70).c-Myc negatively regulates BBC3 (PUMA), ICAM1 (ICAM1) and TP53INP1(TP53INP1) (Amente et al., Nucleic Acids Res, 2011, 39, 9498-9507; Yunet al., Blood, 2016, 127, 2711-2722; Florea et al., PLoS ONE, 2013, 8,e73146). Upregulation of PUMA, ICAM1 and TP53INP1 is also consistentwith the inhibition of c-Myc by PG3-Oc (FIG. 70). GSEA analysisindicated that downregulation of genes was enriched in the c-Myc pathwayand network, suggesting PG3-Oc has significant impact on the c-Mycpathway and network (FIG. 6L).

Example 9: ERK1/2 Mediates PG3-Oc-Induced Degradation of c-Myc

SB216763 is a GSK3α/β inhibitor. It has been reported thatregorafenib-induced and GSK3β-dependent NF-κB (p65) activation mediatesupregulation of PUMA in a p53-independent manner in several colorectalcancer cell lines, and upregulation of PUMA can be inhibited by SB216763(Chen et al., Clin Cancer Res, 2014, 20, 3472-3484). SB216763 did blockPG3-Oc-mediated upregulation of PUMA, but was permissive for thedegradation of c-Myc protein (FIG. 97). Surprisingly, it was found thatPG3-Oc treatment led to activation of AKT and inhibition of GSK3β,indicated by increased inhibitory phosphorylation of GSK3β at Ser9 (FIG.98). Furthermore, knockdown of GSK3β did not have any effect onPG3-Oc-induced upregulation of PUMA (FIG. 99), which is consistent withthat GSK3β was inhibited by PG3-Oc, and the knockdown of NF-κB subunitp65 did not prevent PUMA from induction by PG3-Oc (FIG. 31D). BecauseSB216763 inhibits both GSK3α/β, therefore, both GSK3α/β, which again didnot prevent c-Myc from degradation and PUMA were knocked down frominduction by PG3-Oc (FIG. 100). Taken together, these data suggest thatGSK3β is inhibited by PG3-Oc, and is not involved in the degradation ofc-Myc protein in PG3-Oc treated cells, and SB216763 blunted theinduction of PUMA possibly through off-target inhibition of unknowntargets which are required for c-Myc-downregulation-mediatedupregulation of PUMA.

It was found that PG3-Oc potently blocked phosphorylation of ERK1/2 in atime- and dose-dependent manner in Jurkat cells (FIG. 62). PG3-Octreatment resulted in rapid dephosphorylation of ERK1/2, andsimultaneous dephosphorylation of their direct target, c-Myc, at Ser 62in HT29 cells (FIG. 72), indicating inhibition of ERK1/2. However, thelevels of the phosphorylation of ERK1/2 gradually increased with time(FIG. 72). That is possibly because growth factors in the culture mediumcan stimulate the phosphorylation of ERK1/2. While phosphorylation ofERK1/2 was more evident at 8 (FIG. 72) and 24 hours (FIG. 73), incontrast, the level of phospho-c-Myc at the 8-hour time point wassignificantly decreased, and was undetectable at 24 hours as compared tothe untreated control, suggesting that PG3-Oc also inhibits the functionof phospho-ERK1/2.

Hence, it was hypothesized that inhibition of ERK1/2 by PG3-Oc can leadto the dephosphorylation of c-Myc at Ser62 and subsequent degradation ofc-Myc. To test this hypothesis, two ERK1/2 inhibitors that act bydifferent mechanisms were used. U0126 is an indirect ERK1/2 inhibitor.It inhibits MEK1 kinase and blocks the phosphorylation of ERK1/2.SCH772984 directly binds to ATP-binding pockets of ERK1/2 and inhibitsERK1/2 regardless of the phosphorylation status of ERK1/2, and is apotent and highly selective ERK1/2 inhibitor (Chaikuad et al., Nat ChemBiol, 2014, 10, 853-860). As seen in FIG. 73, both inhibitors potentlyinduced the downregulation of c-Myc and the upregulation of PUMA in HT29cells, which phenocopied PG3-Oc treatment in the degradation of c-Mycand the upregulation of PUMA, indicating that ERK1/2 is an importantmediator in the control of both c-Myc stability and PUMA induction.Consistent with a previous publication (Choi et al., Genes Dev, 2010,24, 1236-1241), SCH772984 can inhibit ERK1/2 function after ERK1/2 isphosphorylated (FIG. 73), and the same is observed for PG3-Oc (FIG. 73).Co-treatments indicated that neither U0126 nor SCH772984 enhancedPG3-Oc-induced upregulation of PUMA compared to PG3-Oc alone (FIG. 73),suggesting that PG3-Oc and SCH772984 share the same target ERK1/2.

EGFR signaling pathway activation can lead to the phosphorylation andthe activation of ERK1/2. Hence, this pathway was chosen as a model tofurther verify this observation, that is, PG3-Oc inhibits phosphorylatedERK1/2. HT29 cells were cultured in charcoal-stripped medium so thatERK1/2 is maintained at a low phosphorylation status due to the removalof growth factors. Pre-treatment of cells with PG3-Oc, SCH772984 andEGFR inhibitor gefitinib respectively, was followed by addition of EGFto activate the EGFR pathway. PG3-Oc treatment resulted in a decrease ofphosphorylation of ERK1/2 and downregulation of c-Myc. Subsequentaddition of EGF stimulated the increase of ERK1/2 phosphorylation, butthis did not rescue c-Myc from degradation (FIG. 74). Same result wasobserved for SCH772984 and it was more potently than PG3-Oc (FIG. 74).These data confirm that PG3-Oc is able to inhibit phospho-ERK1/2. Incontrast, EGFR inhibitor gefitinib did not induce degradation of c-Mycand upregulation of PUMA, and was able to completely blockEGF-stimulated phosphorylation of ERK1/2, suggesting that PG3-Ocinhibits ERK1/2 not through inhibition of EGFR (FIG. 74).

To further confirm the role of inhibition of ERK1/2 in regulating c-Mycand PUMA, siRNA knockdown of ERK1, ERK2 and both ERK1/2 respectively,was performed which led to potent downregulation of c-Myc andupregulation of PUMA (FIG. 75). Taken together, these data suggest thatPG3-Oc inhibits ERK1/2 and leads to degradation of c-Myc andupregulation of PUMA.

Besides PUMA, PG3-Oc treatment also induces upregulation of DR5, p21 andNoxa (FIGS. 28C, 28D, 45, 31A, 28A and 28B). It is interesting to knowwhether SCH772984 also has impact on those p53 target genes. As shown inFIG. 76, SCH772984 did not induce DR5, shown mild upregulation of p21and suppressed Noxa expression. Thus PG3-Oc has unique effects in p53pathway restoration that go beyond effects on ERK1/2 and Puma induction.

Regorafenib is an FDA-approved drug for treatment of metastaticcolorectal cancer, advanced gastrointestinal stromal tumors andhepatocellular carcinoma. Regorafenib inhibits ERK and leads to theinduction of PUMA through the GSK3β/NF-κB axis and induces PUMA-mediatedapoptosis in colon cancer cell lines (Chen et al., Clin Cancer Res,2014, 20, 3472-3484). PG3-Oc was compared with regorafenib in theinduction of PUMA and DR5. Regorafenib (40 μM and 24 hours treatment)was used as a control, as previously reported (Chen et al., Clin CancerRes, 2014, 20, 3472-3484). As shown in FIG. 77, 4 μM PG3-Oc induced amuch higher level of PUMA and DR5 in both HT29 and HCT116 p53^(−/−) celllines at the 24-hour time point compared to 40 μM regorafenib. Inaddition, the level of cleaved caspase-8 was also significantly higherwith PG3-Oc than regorafenib in HCT116 p53^(−/−) cells (FIG. 77) at the24-hour time point.

Example 10: Inhibition of Tumor Growth In Vivo in HT29 Tumor Xenograftsby PG3-Oc

To evaluate the antitumor effects of PG3-Oc in vivo, a human tumorxenograft model was established by subcutaneous injection of human coloncancer cells into nude mice. After the tumor volume reachedapproximately 50 mm³, mice were treated by i.p. injection with vehicleor PG3-Oc at 5 mg/kg 3 times weekly for 2 weeks. With HT29 xenografts,tumor volume in PG3-Oc-treated mice appeared to be significantly reducedas compared with vehicle-treated mice (FIGS. 78, 79 and 80). Ki-67expression was found to be significantly decreased in PG3-Oc-treatedtumors as compared with the vehicle group (FIG. 82). PUMA wassignificantly induced in PG3-Oc-treated tumors as compared with controls(FIG. 82). No significant difference in body weight was observed betweenPG3-Oc and the vehicle treatment groups (FIG. 81). These resultsindicate that PG3-Oc inhibits tumor growth in the HT29 mouse xenograftmodel.

In summary, evidence is provided for a novel prodigiosin analog PG3-Ocwhich has potent anti-tumor activity both in vitro and in vivo in adiverse panel of mutant p53 cancer lines. A model in which inhibition ofERK1/2 by PG3-Oc results in destabilization and degradation c-Myc isproposed, which leads to upregulation of PUMA. PUMA-mediated activationof caspase8 causes cell apoptosis (FIG. 83). PG3-Oc treatment also leadsto upregulation of DR5 through the ATF4/CHOP axis, which sensitizesTRAIL-resistant cells to TRAIL treatment (FIG. 83).

Various modifications of the described subject matter, in addition tothose described herein, will be apparent to those skilled in the artfrom the foregoing description. Such modifications are also intended tofall within the scope of the appended claims. Each reference (including,but not limited to, journal articles, U.S. and non-U.S. patents, patentapplication publications, international patent application publications,gene bank accession numbers, and the like) cited in the presentapplication is incorporated herein by reference in its entirety.

The claims are not limited to the embodiments described and exemplifiedabove, but is capable of variation and modification within the scope ofthe appended claims.

1. A compound of Formula XIV:

wherein: R¹ and R² are, independently, selected from the groupconsisting of H, OH, halogen, —C₁₋₆alkyl, —C₁₋₆fluoroalkyl, —CN, —NO₂,—OR⁷, —SR⁷, —S(═O)R⁷, —S(═O)₂R⁷, —NHS(═O)₂R⁷, —C(═O)R⁷, —OC(═O)R⁷,—CO₂R⁷, —OCO₂R⁷, —CH(R⁷)₂, —N(R⁷)₂, —C(═O)N(R⁷)₂, —NHC(═O)NHR⁷,—NHC(═O)R⁷, —NHC(═O)OR⁷, —C(OH)(R⁷)₂, and —C(NH₂)(R⁷)₂; each R⁷ is,independently, H, halogen, or C₁-C₆alkyl, wherein the alkyl group isoptionally substituted by 1, 2, 3, 4, or 5 substituents independentlyselected from halogen, OH, CN, and NO₂; R³ is an optionally substitutedaryl or an optionally substituted heteroaryl; R⁴, R⁵, and R⁶ are,independently, OH, —C₁₋₁₀alkyl, —OC₁₋₁₀alkyl, or —SC₁₋₁₀alkyl, whereineach alkyl group is, independently, optionally substituted by 1, 2, 3,4, or 5 substituents independently selected from halogen, OH, CN, andNO₂; and n is an integer from 0 to 5; or an isomer, tautomer, or solvatethereof, or a pharmaceutically acceptable salt thereof; provided that:if n is 0, then R³ is not an optionally substituted pyrrolyl; and if nis 2, then the compound is not


2. The compound according to claim 1, or isomer, tautomer, or solvatethereof, or pharmaceutically acceptable salt thereof, wherein R¹ and R²are, independently, selected from the group consisting of H, halogen,—C₁₋₆alkyl, —C₁₋₆fluoroalkyl, —OR⁷, —SR⁷, —S(═O)R⁷, —C(═O)R⁷, —OC(═O)R⁷,—CO₂R⁷, —OCO₂R⁷, —CH(R⁷)₂, —C(═O)N(R⁷)₂, —C(OH)(R⁷)₂, and —C(NH₂)(R⁷)₂.3. The compound according to claim 1, or isomer, tautomer, or solvatethereof, or pharmaceutically acceptable salt thereof, wherein each R⁷is, independently, H, halogen, or C₁-C₆alkyl.
 4. The compound accordingto claim 1, or isomer, tautomer, or solvate thereof, or pharmaceuticallyacceptable salt thereof, wherein R¹ is —OCH₃ and R² is H.
 5. Thecompound according to claim 1, or isomer, tautomer, or solvate thereof,or pharmaceutically acceptable salt thereof, wherein R³ is an optionallysubstituted heteroaryl.
 6. The compound according to claim 1, or isomer,tautomer, or solvate thereof, or pharmaceutically acceptable saltthereof, wherein R³ is an optionally substituted heteroaryl selectedfrom the group consisting of pyridyl, pyrimidinyl, pyrazinyl,pyridazinyl, pyridinyl, triazinyl, furyl, quinolyl, isoquinolyl,thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, pyrazolyl,isoxazolyl, triazolyl, tetrazolyl, indazolyl, isothiazolyl, purinyl,carbazolyl, isoxazolyl, indolinyl, pyranyl, pyrazolyl, triazolyl,oxadiazolyl, thianthrenyl, indolizinyl, isoindolyl, pyrrolyl, andxanthenyl.
 7. The compound according to claim 1, or isomer, tautomer, orsolvate thereof, or pharmaceutically acceptable salt thereof, whereinR⁴, R⁵, and R⁶ are, independently, OH, —C₁₋₆alkyl or —OC₁₋₁₀alkyl,wherein each alkyl group is, independently, optionally substituted by 1,2, or 3 substituents independently selected from halogen, OH, CN, andNO₂.
 8. The compound according to claim 1, or isomer, tautomer, orsolvate thereof, or pharmaceutically acceptable salt thereof, wherein nis an integer from 1 to
 3. 9. The compound according to claim 1, orisomer, tautomer, or solvate thereof, or pharmaceutically acceptablesalt thereof, wherein: R¹ and R² are, independently, selected from thegroup consisting of H, halogen, —C₁₋₆alkyl, —C₁₋₆fluoroalkyl, —OR⁷,—SR⁷, —S(═O)R⁷, —C(═O)R⁷, —OC(═O)R⁷, —CO₂R⁷, —OCO₂R⁷, —CH(R⁷)₂,—C(═O)N(R⁷)₂, —C(OH)(R⁷)₂, and —C(NH₂)(R⁷)₂; each R⁷ is, independently,H, halogen, or C₁-C₆alkyl; R³ is an optionally substituted heteroaryl;R⁴, R⁵, and R⁶ are, independently, OH, —C₁₋₆alkyl or —OC₁₋₁₀alkyl,wherein each alkyl group is, independently, optionally substituted by 1,2, or 3 substituents independently selected from halogen, OH, CN, andNO₂; and n is an integer from 1 to 3; provided that: if n is 2, then thecompound is not


10. The compound according to claim 1, or isomer, tautomer, or solvatethereof, or pharmaceutically acceptable salt thereof, wherein: R¹ and R²are, independently, selected from the group consisting of H, halogen,—C₁₋₆alkyl, —OR⁷, —SR⁷, —S(═O)R⁷, —C(═O)R⁷, —OC(═O)R⁷, —CO₂R⁷, —OCO₂R⁷,—CH(R⁷)₂, and —C(OH)(R⁷)₂; each R⁷ is, independently, H, halogen, orC₁-C₃alkyl; R³ is an optionally substituted heteroaryl selected from thegroup consisting of pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,pyridinyl, furyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl,oxazolyl, pyrazolyl, isoxazolyl, indazolyl, isothiazolyl, purinyl,carbazolyl, isoxazolyl, indolinyl, pyranyl, pyrazolyl, oxadiazolyl, andpyrrolyl; R⁴, R⁵, and R⁶ are, independently, OH, —C₁₋₆alkyl or—OC₁₋₈alkyl, wherein each alkyl group is, independently, optionallysubstituted by 1 or 2 substituents independently selected from halogenand OH; and n is an integer from 1 to 3; provided that: if n is 2, thenthe compound is not


11. The compound according to claim 1, or isomer, tautomer, or solvatethereof, or pharmaceutically acceptable salt thereof, wherein: R¹ and R²are, independently, selected from the group consisting of H, —C₁₋₆alkyl,—OR⁷, —C(═O)R⁷, and —OC(═O)R⁷; each R⁷ is, independently, halogen orC₁-C₃alkyl; R³ is an optionally substituted heteroaryl selected from thegroup consisting of pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,pyridinyl, furyl, thienyl, imidazolyl, indolyl, pyrryl, purinyl,pyranyl, pyrazolyl, oxadiazolyl, and pyrrolyl; R⁴ and R⁶ are,independently, —C₁₋₃alkyl, and R⁵ is OH or —OC₁₋₈alkyl, wherein eachalkyl group is, independently, optionally substituted by 1 or 2halogens; and n is 2 or 3; provided that: if n is 2, then the compoundis not


12. The compound according to claim 1, or isomer, tautomer, or solvatethereof, or pharmaceutically acceptable salt thereof, wherein: R¹ and R²are, independently, selected from the group consisting of H, —C₁₋₆alkyl,and —OR⁷; each R⁷ is, independently, halogen or C₁-C₃alkyl; R³ is anoptionally substituted heteroaryl selected from the group consisting ofpyridyl, pyrimidinyl, pyridinyl, imidazolyl, indolyl, pyrryl, purinyl,pyranyl, and pyrrolyl; R⁴ and R⁶ are, independently, —C₁₋₃alkyl; R⁵ isOH or —OC₆₋₈alkyl; and n is 2 or 3; provided that: if n is 2, then thecompound is not


13. The compound according to claim 1, or isomer, tautomer, or solvatethereof, or pharmaceutically acceptable salt thereof, wherein: R¹ is—OR⁷, wherein R¹ is halogen or C₁-C₃alkyl; R² is H; R³ is an optionallysubstituted heteroaryl selected from the group consisting of pyridyl,pyrimidinyl, pyridinyl, pyrryl, and pyrrolyl; R⁴ and R⁶ are,independently, —C₁₋₃alkyl; R⁵ is —OC₆₋₈alkyl; and n is 2; provided that:the compound is not


14. A pharmaceutical composition comprising the compound according toclaim 1, or an isomer, tautomer, or solvate thereof, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 15. The pharmaceutical composition according toclaim 14, further comprising an anti-cancer agent.
 16. A method oftreating cancer in a subject comprising administering to the subject inneed thereof the compound, or isomer, tautomer, or solvate thereof, orpharmaceutically acceptable salt thereof, according to claim
 1. 17. Themethod according to claim 16, wherein the cancer is a colorectal cancer,a head and neck cancer, a pancreatic cancer, a breast cancer, a coloncancer, a lung cancer, or a glioblastoma cancer.
 18. The methodaccording to claim 16, further comprising administering to the subjectanother anti-cancer therapy.
 19. The method according to claim 18,wherein the another anti-cancer therapy is radiation therapy,chemotherapy, or immunotherapy, or any combination thereof. 20.(canceled)